This paper reviews recent advances in graphene-based biosensors development in order to obtain smaller and more portable devices with better performance for earlier cancer detection. In fact, the potential of Graphene for sensitive detection and chemical/biological free-label applications results from its exceptional physicochemical properties such as high electrical and thermal conductivity, aspect-ratio, optical transparency and remarkable mechanical and chemical stability. Herein we start by providing a general overview of the types of graphene and its derivatives, briefly describing the synthesis procedure and main properties. It follows the reference to different routes to engineer the graphene surface for sensing applications with organic biomolecules and nanoparticles for the development of advanced biosensing platforms able to detect/quantify the characteristic cancer biomolecules in biological fluids or overexpressed on cancerous cells surface with elevated sensitivity, selectivity and stability. We then describe the application of graphene in optical imaging methods such as photoluminescence and Raman imaging, electrochemical sensors for enzymatic biosensing, DNA sensing, and immunosensing. The bioquantification of cancer biomarkers and cells is finally discussed, particularly electrochemical methods such as voltammetry and amperometry which are generally adopted transducing techniques for the development of graphene based sensors for biosensing due to their simplicity, high sensitivity and low-cost. To close, we discuss the major challenges that graphene based biosensors must overcome in order to reach the necessary standards for the early detection of cancer biomarkers by providing reliable information about the patient disease stage. The cresponse . The aspired goal in an accurate and early-stage diagnosis of cancer is based on the detection and quantification of reliable cancer biomarkers by cost-effective and less invasive methods. Researchers believe that early diagnosis of diseases with minimal cost and time-consumption may become achievable due to recent advances in the development of biosensors. These devices use biorecognition elements for the selective interaction with an analyte and different types of transducers to obtain the signal readout. The operational characteristics of biosensors have been reported as improving substantially when nanomaterials such as graphene and its derivates are employed. Graphene is aromatic, hydrophobic and chemical inert; moreover, it is biocompatible and has the facility to adsorb biomolecules due to π-π stacking between its hexagonal cells and the carbon rings present in the majority of nano/biomolecules. Owing to its exceptional charge mobility and atomic thickness, graphene have been proposed as the basis to sensitive detection and chemical/biological free-label ,6. FurthHerein we intent to discuss the most relevant graphene based technology for the development of different types of biosensors skillful to detect characteristic cancer biomolecules in early stages or overexpressed on cancerous cells surface. We summarize the current existing challenges on the cancer biomarker detection and quantification through graphene based biosensors in order to increase the selectivity and sensitivity. Finally, we intent to give a global view about the challenges for future development of various types of graphene based biosensors for cancer detection.2-hybridized carbon bonds in aromatic structure. Although at the moment several synthetic methodologies allow to obtain this material, most of them are really far from provide the theoretical structure, producing instead graphene sheets with different chemical or/and physical defects. The most common defects observed on “graphene” are: sheets with different numbers of atomic layers that can go from monolayer until multilayer; the atomic flatness is not real because the sheets tend to distort due to the presence of structural defects such as lattice missing carbon atoms or inclusion of other foreign atoms, in particular oxygen; and different lateral dimensions of the sheets [Theoretically, graphene sheets are perfect 2D single crystal formed by spe sheets . These ie sheets . In ordee sheets . This stHereafter we describe the family of graphene and its derivatives , their mChemical vapor deposition (CVD) production of graphene offers the possibility to obtain large sized films that are ideally for the fabrication of electronic sensor devices with high specific detection area and low noise . HoweverThe synthesis of large graphene sheets is also possible through the epitaxial growth on the basal plane of single-crystal silicon carbide. The big advantage of this approach is that graphene films formed at silicon carbide (SiC) surface can be directly used for sensor development since the substrate is a good insulator and the strong interaction between graphene and SiC creates a graphene bandgap to 0.26 eV, which is ideal for sensors whose detection is based in induced field effect . However3 C-C bonds [2 lattice and residual oxygen contents (dependent of the reducing method applied). Nevertheless, rGO is considered the most promising material for electrochemical sensors, since it is electrochemically more active than any other graphene family member [The wet chemical exfoliation of graphite is a very common adopted approach for the synthesis of GO in large scale and at low cost. GO is an electrical insulator, chemically heterogeneous with high amount of oxygen groups linked to the carbon lattice forming defect regions with sp-C bonds . GO has -C bonds . Further-C bonds . The resy member . Furthery member . The pioy member . They dey member .Graphene quantum dots (GQDs), also referred as Nano-graphene (NG) are nanometer-sized fragments with distinct properties from graphene, which makes them interesting candidates for a whole range of new applications. The methods to synthesize NG can be classified into two types: the top-down and bottom-up methods  being thet al. [The top-down approaches include hydrothermal , electroet al.  reportedet al.  and alsoet al. . Indeed,et al.  which diet al. .Nano-graphene oxide (NGO) is a new class of carbon based materials that was proposed for biomedical applications due to its small size, intrinsic optical properties, large specific surface area, and easy to functionalize . The preOne very simple way to induce further sensing capabilities to graphene is by changing its physical-chemical structure. The changes required to obtain new graphene features can be made by the chemical doping of its structure with different foreign atoms that promote changes in electrical, electrochemical and optical properties. The most typical methodologies for atomic modifications of graphene surface includes oxygenation, hydrogenation and fluorination by using different synthetic approaches . Another2 hybridized carbon bonds of graphene and/or the formation of covalent bonds  between the functional molecule and the oxygen groups at graphene surface [There are two main different approaches for the surface functionalization of graphene based materials: covalent and noncovalent. The covalent functionalization is very straightforward when using as a precursor GO sheets. The presence of oxygen functional groups like carboxylic or hydroxyl at GO surface enables the establishment of covalent bonds with other molecules, polymers, biological structures and nanoparticles. The range of chemical mechanism available to design new covalent bonds between graphene and other organic entities is very broaden following the basic principles of organic chemistry . The org surface . For exa surface . Many ot surface . In gene surface . This fe surface . Further surface .2-hybridized carbon network. That fact offers a number of unique physicochemical properties that are desirable and advantageous for sensing applications. Pristine graphene possesses an extensive aromatic surface that allows the molecular interaction via π-π stacking and due to the atomic thickness the electric, mechanic and optical properties are highly sensitive to adsorbed molecules. Recently, the optoelectronic properties of graphene were explored as a biosensor for chemically specific label-free detection of proteins [In case of graphene, although covalent strategies can be more efficient and stable they request dramatically changes on the structure by the disruption of the C=C bonds leading to the alteration of its characteristic electronic properties. For this reason, non-covalent functionalization of graphene is considered a simple and versatile method, offering the opportunity to attach different functionalities while simultaneously maintaining the integrity of the C-C spproteins . The eleproteins . For exaproteins . 2/sp3 carbon regions on a global network [3 gases at room temperature in comparison to that of pure rGO [Reduced Graphene Oxide as explained before results from the partial recovery of aromatic structure through the gradual reduction of GO, showing an hybrid structure between sp network . rGO off network , molecul network  and gas  network . Despitepure rGO . The enhpure rGO .ex situ) [The closest integration of nanoparticles on graphene based materials surface are of a particular interest because it offer the possibility to obtain the individual properties of each element in a single material and at same time exhibit additional synergistic effects that can be very important on sensor development through the development of new sensing mechanism or increasing the sensitivity and selectivity of the system. The functionalization of graphene, GO and rGO with nanoparticles can occur by both covalent and noncovalent interactions using many different synthetic routes: growing the nanoparticles on their surface (in situ) or by attaching pre-synthesized nanoparticles to the graphene surface (ex situ) . Howeverex situ) . The majex situ) . Nanoparex situ) . The greIn a near future, biosensors are expected to detect with high sensitivity and selectivity a wide range of cancer biomarkers present in body fluids or overexpressed on cancer cells surface in order to diagnosis and monitoring the evolution of the disease during the treatment. In this way, biosensing systems employing the properties of graphene derivatives for cancer detection have found their way into a wide variety of strategies capable of improving the sensing efficiency of cancer biomarkers. In the following sections, we describe the application of graphene in optical imaging methods such as photoluminescence and Raman imaging, electrochemical sensors for enzymatic biosensing, DNA sensing, and immunosensing. The changes that occur in the DNA sequence of the genomes of cancer cells are the reason of cancers arising . The proThe possible chemical interactions of this type of powerful molecular probes or steric hindrance with biomolecules may cause biotoxicity or perturbation to the systems being investigated which effect is not conceivable in medical applications .The development of nanoparticles with multifunctionalities has been a research focus in the last decade including graphene and its derivatives . The unpet al. [The high contrast bio-imaging produced by NG in the detection of cancer cells was demonstrated in the work of Peng et al.  The greeet al. [The photoluminescence of GO was first used for cellular imaging by Dai et al.  NGO was In vivo studies using NG prepared by a simple hydrothermal method with polythiophenes as the precursors indicated that NG can act as a multifunctional nanoplatform for the simultaneous imaging and highly efficient in photodynamic therapy (PDT) of cancer [et al. [f cancer . Tumor-tf cancer  and hyalf cancer  could be [et al.  developeet al. [in vivo biodistribution and tumor specific delivery of NG-HA were also studied by imaging fluorescence. To investigate the efficient delivery of NG, the authors have used balb/c mice with 6 weeks of age. The tumors were grown, during 10 days (volume of 100mm3) on the back of the mice using A549 cells. to tumor tissue and its biodistribution, NG-HA was injected intravenously (10 mg/kg of body weight) through the tail vein. After 2 h, the mice with tumors were sacrificed and their organs dissected. The strong fluorescence from the tumors indicates the targeted accumulation of NG-HA to the tumor site  to form (AbA5)-modified NG (AbA5-NG) enabling to label apoptotic cells in live zebrafish (Danio rerio) [−1) were separately recorded. It was possible to observe that the photoluminescent spots were distributed around the cell membranes and cytoplasm suggesting that NG penetrated into the cells via receptor-mediated and/or nonreceptor-mediated endocytosis. This study opens a novel opportunity to visualize the initiation and progression of apoptosis which might help in understanding the apoptotic mechanism better which is of major importance in diagnosis and treatment of cancer.Recently, Roy o rerio) . Zebrafiin vivo imaging of cancer with biocompatible surface-enhanced Raman scattering (SERS) probes have emerged although its application is traditionally in vitro. Fluorescence imaging is the most common bioimaging technique [In the past few years the first reports of the feasibility of echnique ,70,71 foechnique ,73. In cechnique ,75. The echnique . This alechnique ,78,79. Graphene oxide is an excellent candidate for biomedical imaging due to its inherent fluorescence and Raman scattering activity . GO and GO decorated with Au nanoparticles were internalized into individual cells to provide localized sensing at the subcellular level . The senet al. [The study of the mechanism of the cellular uptake was also performed by Huang et al. , using Get al. have proposed several SERS substrates based on the nucleation of gold nanohexagons (Au) on graphene nanosheets [Manikandan nosheets . These wet al. [A gold capture substrate modified with linker molecules and antibodies, covered by a graphene monolayer, was developed by Krasnoslobodtsev et al. . In thiset al. . A SERS-et al. . In thiset al. [Based on the natural high affinity of folate for the folate receptor protein (FR), which is commonly expressed on the surface of many human cancers, folate conjugates bind to the FR and causes the cellular uptake via endocytosis. The conjugation of the specific binding of folic acid (FA) and graphene-metal nanoparticles improve the targeted detection of cancer cells. Liu et al.  preparedet al.  by usingIn order to apply SERS in real-world, the nucleosides excreted from normal human urine (n = 40) and from breast cancer patients (n = 40) were measured using a graphene-silver-copper nanoparticles (G/SCNP) as SERS probe . On the in situ inside HeLa cells. With such results it was clearly demonstrated that SERS offers distinct advantages over the widely used fluorescence technique in that it can provide direct information on the carrier and the drug inside the living cells simultaneously, and without dye labeling.The chemotherapy is the most common cancer treatment but its huge side-effects make it very painful for the patients. It is urgent to promote effective and targeted deliver of drugs used in chemotherapy since they show good results. SERS can contribute to monitor such deliver using multifunctional nanoprobes. GO-based nanoplatform simultaneously loaded with chemical drug and Ag nanoparticles was prepared and employed to study the drug release from GO in living cancer model cells by SERS . In thisA biosensor is defined as an integrated device that is able of provides quantitative or semi-quantitative information about biomolecules or biostructures by using biological recognition element. The main difference relatively of a bioanalytical system is that requires additional processing steps such as addition of reagents in order to be able to quantify the desired analyte. The structure of a sensor is usually constituted by two key elements: the receptor and the transducer. The receptor should guaranty the specific interaction with the proper analytic or many different analytes and the transducer is responsible to convert the perturbation caused on the receptor (input) through the interaction with the analyte into a measurable signal (output).Hereafter we will discuss particularly electrochemical methods such as voltammetry and amperometry which are generally adopted transducing techniques for the development of graphene based sensors for biosensing due to their simplicity, high sensitivity and low-cost. However sensors based on other detection methods like FRET, photoluminescence, fluorescence, optical and SERS start to arise with very promising results on the biodetection of cancer markers.Immunoassay has becoming one of the most important tools for clinical diagnosis of cancer. Detection of cancer biomarkers associated with certain tumors in early stage of the disease becomes on important contribute for the successful treatment. Graphene sensors promise to be an innovative technology in next generation electronics on cancer diagnostic applications, due to exceptional electronic properties and extreme surface to volume ratio able to offer greatly enhanced sensitivity for the detection of cancer biomarkers. New graphene-based technology allows the development of portable and rapid diagnostic sensors whit potential to overcome the features of conventional enzyme-linked immunobsorbant assays (ELISAs). Hereafter we describe the potential use of graphene and its derivatives on the development of new electrochemical sensors for detection of specific cancer biomarkers.−1 with the detection limit as 0.03 ng·mL−1. Another work reported the development of a novel electrochemical immunosensor based on the deposition of ultrathin Au-Pt nanowire-decorated thionine/reduced graphene oxide (AuPtNWs/THI/rGO) on the surface of glass carbon electrode (GCE) [−1 to 100 ng·mL−1 with an detection of 6 fg·mL−1. A very simple approach using GO-Thi-Au nanocomposites as sensing platform for the surface modification of GCE was been successfully employed for the fabrication of ultrasensitive electrochemical immunosensor for CEA [−1 to 1 × 109 fg·mL−1 and ultralow detection limit of 0.05 fg·mL−1.Carcinoembryonic antigen (CEA) is one of the most extensively used tumor markers on early detection of lung, ovarian carcinoma, breast cancers and cystadenocarcinoma. A novel electrochemical immunosensor based on carbon ionic liquid electrode (CILE) modified with AuNPs, rGO and poly(l-Arginine) nanocomposite material as sensitive platform for detection of CEA . The synde (GCE) . The dif for CEA . The immet al. reported the Au electrode surface modification by deposition of a thin film of silver and then with a AuNPs-rGO-chitosan nanocomposite prepared by cryogel functionalized with anti-CEA [−1 with a detection limit of 0.1 fg·mL−1. In another work Au electrode surface was initially modified with rGO-SWCNTs-Chitosan nanocomposite (Chi-rGO-CNTs) followed by electrodeposition with Au and Pt NPs and then functionalized with anti-CEA. The proposed immunosensor showed CEA determination of range 0.1 pg·mL−1 to 40 ng·mL−1 with a limit of detection down to 30 fg·mL−1 [et al. reported for the first time a new graphene paper electrode based on poly:poly(styrenesulfonate)(PEDOT:PSS)/rGO composite as a promising alternative over the expensive conventional electrodes  [−1·mL·cm−2 in the detection linear range of 2–8 ng·mL−1. Electrochemical immunosensors based on Au electrodes modified with graphene nanocomposites as a sensing platform showed to be also a viable approach for the detention of CEA. Samanman anti-CEA . At opti fg·mL−1 . Recentland GCE) . rGO PEDin situ generated hydrogen peroxide as coreactant (ZnONPs@rGO-GOD-Ab2) [in situ generating hydrogen peroxide with glucose oxidase and the catalysis of ZnONPs to the ECL reaction of luminol–H2O2 system. The rGO-based immunosensor exhibited a detection range of CEA from 10 pg·mL−1 to 80 ng·mL−1 and a detection limit of 0.0033 ng·mL−1. Recently, another work describes the use of rGO on both sensor sensing platform and as a signal amplifying factor for construction of the “sandwich-type” immunosensor with ultrahigh efficiency for CEA detection [5 pg·mL−1 and a limit of detection 8 pg·mL−1. Graphene biosensors based on sandwich-type immunosensors methods were also been extensively explored on the detection of CEA. High-affinity antibodies and appropriate labels are usually employed for the amplification of detectable signal . Recent GOD-Ab2) . The senetection . Graphen2O2 as an enzyme substrate, in a sandwich-type immunoassay format. Experimental results showed linear relationship between peak current and CEA concentration for the range of 5–60 ng·mL−1. The limit of detection was 5 ng·mL−1. Several works in the last year have been reported proposing new architectures for the development of sandwich-type graphene-based electrochemical immunosensor for CEA detection, where graphene can acts as sensing platform at electrode surface, label agent for the increase of sensitivity or by performing both activities. Numerous studies in the last year have been reported proposing new architectures for the development of sandwich-type graphene-based electrochemical immunosensor for CEA detection, where graphene can acts as sensing platform at electrode surface, conjugate agent for the increase of sensitivity or perform both activities. The performance of each sandwich-type graphene-based biosensor was compared in Graphene made by CVD was also explored as electrochemical sensing platform, in the form of high surface area conducting electrodes surfaces for the high sensitive detection of CEA. The graphene sensing surface was modified with magnetic beads (MBs) functionalized with CEA detection antibody (Gr/MBs-Ab1), by applying an external magnetic field. In order to enhance the sensitivity of the sensor, AuNPs were modified with horse radish peroxidase (HRP) and detection antibody (Ab2), to form the conjugate Ab2–AuNPs–HRP . The ele−1 with a low detection limit of 0.01 ng·mL−1. Recently a new approach based on highly conductive crumpled like structure rGO/AuNPs nanocomposite as a sensing platform for detection of PSA was reported [−1 and the detection limit 0.59 ng·mL−1. Prostate specific antigen (PSA) is a characteristic tumor marker of prostate cancer used in prostate cancer diagnosis and screening. The first attempt to use graphene for sensitive detection of PSA was trough the fabrication of a label-free electrochemical immunosensor. The sensor technology was based on the surface functionalization of GCE electrode with a thin film of the nanocomposite, rGO, 1-pyrenebutanoic acid, succinimidyl ester (PBSE) and CoNPs, as a sensing platform . The PBSreported . The 3D 2-MCM48), able to improve the electron transfer ability of the immunosensor system. The workability of immunosensors system Ag@NH2-MCM48/Ab2 and NH2-GS@FCA was based on the linear catalytic current increase with the PSA concentration. The immunosensor showed a good relationship between the current responses toward PSA concentration on the range from 0.01 to 10.0 ng·mL−1 similar to the non-labeled ones, however the detection limit was as much lower 0.002 ng·mL−1. Recently a novel label electrochemical graphene-based immunosensor for PSA was reported. The sensing surface of the GCE was constructed based on the deposition of new amino-functionalized graphene sheet ferrocenecarboxaldehyde composite material (NH2-rGO@FCA) . The senet al. proposed a novel sandwich-type graphene-based electrochemical immunosensor for the detection of CA19-9 [−1, with low detection limit of 0.006 U·mL−1. Carbohydrate Antigen 19-9 (CA 19-9) is not sensitive or specific to use as a screening test for cancer but can be used as a tumor marker if is produced in abnormal amounts. When CA 19-9 level is elevated to high concentrations it is often correlated to with gastrointestinal malignancies such as cholangiocarcinoma, pancreatic cancer, or colon cancer. Yang f CA19-9 . The sen2 functionalized with Cd2+ for increase the electron transfer rate, followed by the immobilization of the antibody CA15-3 (Ab2), resulting in the following sequence (Ab(2)-f-TiO2-Cd2+). The GCE sensing platform was developed by immobilization of rGO functionalized with ionic liquid and primary CA15-3 antibody (Ab(1)) resulting in the sequence GS-Ab(1). Through biorecognition reaction, the target protein CA15-3 was sandwiched between the primary antibody GS-Ab1 and the prepared (Ab2-f-TiO2-Cd2+) bioconjugates, resulting in an enhanced detectable signal. The resultant immumosensor displayed a wide range of linear response (0.02–60 U/mL), ultra-low detection limit (0.008 U/mL), good reproducibility, selectivity and stability towards CA15-3. Carbohydrate Antigen 15-3 (CA 15-3) is a tumor marker used to monitor certain cancers, particularly breast cancer. The first technological approach for the sensitive detection of CA 15-3 using graphene based materials consisted on the development of label-free electrochemical immunosensor . The imm2O2  . The sanet al. developed a simple and reusable strategy for detecting the VEGF concentration in human serum using a graphene based biosensor [3O4NPs functionalized with Avastin, as the specific biorecognition element for VEGF. The amperometric signal was measured by differential pulse voltammetry to quantify the VEGF concentration, being the linear response of the biosensensor observed for the range of VEGF concentrations from 0.03125 to 2 ng·mL−1. The biosensors showed a good stability  and a limit of detection of 0.03125 ng·mL−1.Vascular endothelial growth factor (VEGF) is an important growth factor that regulates the progression of angiogenesis. VEGF overexpression appears in many human cancers, such as brain tumors, lung cancer, breast cancer, gastrointestinal cancer and urinary tract tumors . Lin et iosensor . The sen3O4@Au composites (GMPs). The combination of AFP with the two labels results in sandwich-type immunocomplexe PDDA-G@Lu-Au~HRP-Ab2/AFP/Ab1~GMP. The magnetic sandwich-type immunocomplexes were immobilized on the working screen-printed carbon electrode (SPCEs) by applying a magnetic field. That approach avoids some adverse effects obtained through the surface functionalization of the electrodes. Under the optimized conditions, the ECL method shows a linear range of AFP detection from 0.002 to 20 ng·mL−1 and a low detection limit of 0.2 pg·mL−1. Recently Zhuo et al. proposed the development of a nobel electrochemiluminescent immunosensor based on a new enhancer of peroxydisulfate system for the detection of AFP [−1 and detection of limit of 0.3 pg·mL−1. Alpha-fetoprotein (AFP) is a glycoprotein of fetal component produced during the embryonic period. However, high AFP serum levels can be found mainly in patients with hepatocellular carcinoma or other types of cancer such as, lung cancer, biliary cancer, gastric cancer, pancreatic cancer, teratocarcinoma of the testis. Graphene-based materials have been explored as a sensing platform for the development of noble electrochemical immunoassay in order to amplify the sensitivity on detection of AFP. The pioneer work reported a graphene-based ultrasensitive multiplexed electrochemiluminescent (ECL) immunoassay method for the detection AFP tumor marker . The gran of AFP . The sen−1, good specificity and large linear detection range from 1 pg·mL−1 to 1 μg·mL−1. This SCG label-free lung cancer biosensor showed to have the lower detection limits ever reported for the three different tumors markers ANXA2, VEGF, and ENO1. Graphene is starting to being explored as an effective platform for electrochemical multiplex detection of actively targeted biomarkers, which has clinical relevance. Recently it was reported the development of a suspended single crystalline graphene (SCG) biosensor for multiplex lung cancer tumor markers detection . After t−1 of DNA target. In another work, Wang et al. proposed a novel sensitive and selective electrochemical DNA graphene-based sensor for the detection of BCR/ABL fusion gene in chronic myelogenous leukemia (CML) [DNA sensors are common used approach for early cancer diagnosis and mutation detection. Electrochemical sensors are one of most used approaches on DNA technology development, because DNA bases are electroactive which allows direct electrochemical signals recording during the electrooxidation process. Graphene demonstrate superior electrochemical performance for oxidation of DNA bases due to its outstanding electrochemical properties and large surface-to-volume ratio, that play a very important role on the development of new electrodes with increased sensitivity and simultaneous detection . Electroia (CML) . CML is ia (CML) . The proet al. reported a smart multilayer amperometric sensor based on deposition of rGO onto an ITO electrode, followed by the functionalization with Prussian blue as H2O2 catalyst and laminin matrix protein to promote cell adhesion  over-expressing folate receptors. The increases number of HeLa cells immobilized at electrode surface promotes a formation of insulating layer inducing the reduction of the recorded intensity of the electrochemical signals. This electrode provides a new sensing alternative for cancer cells detection, with high simplicity related with the synthetic process when compared with the previous one, however it showed higher LOD of 250 cells·mL−1 [et al. develop a reusable electrochemical graphene-based sensor using aptamer clinical trial II AS1411 for label-free cancer cell detection [−1 for SKOV-3 cells. Recently scientists started to give attention to the development new graphene ultrasensitive sensors able to quantify cells that can growth directly on its sensing surface or that can specifically detect cancer cells from a very complex mixture. Wu tibodies . This bills·mL−1 . Feng etetection . Graphenetection . The GO et al. developed a new ZnO/graphene composite modified with S6 aptamer as sensitive photoelectrochemical (PEC) strategy for the specific detection of SK-BR-3 cancer cells [−1. Yan et al. reported a novel graphene immunosensor for detection of cancer cells MCF-7 though the surface modification of the working electrode with GO/AuNPs nanocomposite [−1. Another work reported the use of small bifunctional composite quantum dot (Fe3O4/CdSe QD), with intense electrochemiluminescence (ECL) and excellent magnetic property, as reported as enhancer signal probe for sensitive detection of cancer cells via DNA cyclic amplification technique using GO sensing platform to immobilize DNA capture probes (c-DNA1) at the surface of Au electrode [−1 with a detection limit of 98 cells·mL−1. Recently, graphene/inorganic nanoparticles hybrids started to be used as a sensing platform at electrodes surface in order to increase the sensitivity of the sensor. Liu er cells . This hyomposite . The comlectrode . The incGraphene as we discuss before was characterized to process very interesting properties, in particular high conductivity and luminescence quenching ability. In fact, the ultra-high quenching efficiency is attributed to three possible mechanisms: Forter resonance energy transfer (FRET), surface energy transfer (SET) and photo-induced electron transfer . As a quin situ detection of integrin αvβ3. Integrin have a vital role in cancer cell adhesion, proliferation, migration and metastasis [2 aromatic domains of GO to establish noncovalent interactions with pyrene molecules resulting in the quenching effect via fluorescence resonance energy transfer or dipole–dipole coupling effects. On this work GO based biosensor system was initially at a quenching state due to the proximity of RGD–pyrene to GO upon π-π stacking interactions [in situ detection of integrin overexpression on cancer cells surface (MDA-MB-435 cell line). Graphene oxide (GO) based fluorescent biosensor was prepared for real-time tastasis . The bioractions . Howeverin situ detection of tumor cells was also reported [1 to 2.5 × 104 cells·mL−1 and a detection limit about 25 cells·mL−1. Another work suggested a new graphene based fluorescent biosensor system based on a development of a novel molecular aptamer beacon (MA) as fluorescently labelled and GO as the acceptor for target detection by employing long range resonance energy transfer (LrRET) of cellular prion protein (PrPC) [−1 with a detection limit of 0.309 µg·mL−1. A multiplex microfluidic chip integrated with the GO-based FRET strategy to create a screening assay for reported . For thin (PrPC) . InitialTo summarize, we have highlighted the potential of graphene and its derivatives for detection and quantification of cancer biomolecules and cells. Furthermore, we also described numerous procedures capable of convert graphene via surface engineering into specific platforms for particular types of biomolecules or bioreceptors overexpressed by cancerous cells. The biosensing of cancer was discussed giving light to the synergistic properties of the graphene-based sensing entities that can simultaneously act as imaging and therapeutic agents. Additionally, the bioquantification of cancer biomarkers and cells was fully described giving particular attention to specific biomarkers responsible for well-known cancer types.In vitro and in vivo studies have reported that the cytotoxicity of graphene and its derivatives depend on many factors, including preparation protocol, purification processes and final physicochemical properties [Although the great potentialities of graphene in biosensing technologies, its use could present some health concern issues since a large number of toxicological studies presented contradictory conclusions ,133. In operties . Thus, tThe scalable, controllable and reproducible methods of synthesis of graphene-based nanomaterials are other relevant challenges that need to be improved in order to obtain consistent results. Overall, cancer is a disease with high innate heterogeneity, very difficult to detect and diagnose via a single biomarker with high specificity and sensitivity. The detection of cancer biomarker in real biological samples such as blood plasma, blood serum cerebrospinal fluid, saliva or urine is a real challenge since these samples are very complex systems with different kinds of proteins, ions and other chemical species able to promote false positive responses in label free assays. Possible ways to overcome this issue are the passivation of the sensor’s surface with an antifouling agent or, more particularly, the previous filtration/purification of the biological sample in order to concentrate the target entity.Thus, as it was discussed, the ability of graphene based materials to conjugate their outstanding chemical and physical features into remarkable sensing properties appears to be a solid milestone on the long pathway to achieve an early and optimal cancer diagnostics. Indeed, researchers around the world are continuing to explore the wide range of sensing strategies opened by graphene in the last few years due to its proven adaptability and functionality in different cancer microenvironments. For the near future, the ultimate goal is to develop graphene based devices capable of simultaneously detect multiple cancer biomarkers.


Purpose. To introduce a minimally invasive procedure, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), to obtain a pathologic evidence of a definite ocular sarcoidosis in a patient who initially presented with presumed ocular sarcoidosis with pulmonary involvement. Methods. An EBUS-TBNA procedure was performed at perihilar lymph nodes, subcarina, and right paratrachea of the patient and specimen obtained was sent for histocytopathological studies. Result. Histocytopathological findings revealed aggregates of epithelioid histiocytes forming a noncaseous granuloma, a hallmark of sarcoidosis. Conclusion. EBUS-TBNA should be considered an alternative procedure to provide cytohistopathology proven diagnosis of definite ocular sarcoidosis. The diagnosis of sarcoidosis can be reliably established when there is a compatible clinical/radiological picture together with the hallmark histologic findings of noncaseating epithelioid cell granulomas. Sarcoidosis affects predominantly the lungs and thoracic lymph nodes, skin, and eyes. The most predominant finding is peribronchial thickening or bilateral lower lung involvement, especially hilar adenopathy, which occurs in patients with pulmonary sarcoidosis . Ocular A 25-year-old female complained of blurring vision and noticed several small floaters in the right eye of about one month's duration. Initial ocular examination revealed best corrected distant visual acuity of 20/40 in the right eye and 20/20 in the left. Slit lamp biomicroscopy revealed fine mutton fat KPs and trace cells in the anterior chamber bilaterally. Dilated fundus examination disclosed blurred disc margin in both eyes. Multiple vitreous clumping and small yellow spots of retinitis with deposits along the venous wall were seen at the inferior retina, more intense in the right eye than in the left Figures . No exteP < 0.001) [P < 0.001) [For patients with suspected sarcoid uveitis, rationale to use immunologic evidence to diagnose active lesion of sarcoid in the eye is limited. Though recent study reported a high CD4/CD8 ratio of lymphocytes obtained from diagnostic vitrectomy using flow cytometric analysis which delivered a high diagnostic value; such evidence provided the diagnosis of presumed ocular sarcoidosis . A diagn< 0.001) . Another< 0.001) . A recen< 0.001) . HoweverWe demonstrated the role of EBUS-TBNA as an alternative procedure in labeling a diagnosis of definite ocular sarcoidosis and pulmonary sarcoidosis in a young patient who initially presented with presumed ocular sarcoidosis.


Informal peer learning is a particularly powerful form of learning for medical teachers, although it does not always occur automatically in the departments of medical schools. In this article, the authors explore the role of teacher communities in enhancing informal peer learning among undergraduate medical teachers. Teacher communities are groups of teachers who voluntarily gather on a regular basis to develop and share knowledge. Outside of medical education, these informal teacher communities have proved to be an effective means of enhancing peer learning of academic teachers. The processes underlying this outcome are, however, not known. This study therefore aims to explore the processes that make informal teacher communities effective in supporting peer learning of teachers.A qualitative study was performed at a Dutch medical school, where a student-centred undergraduate curriculum had recently been introduced. As part of this curriculum, tutors are segregated into separate specialty areas and thus have only limited opportunities for informal learning with other tutors. The authors followed two informal teacher communities aimed at supporting these tutors. They observed the interactions within the teacher communities and held semi-structured interviews with ten of the participants. The observation notes and interview data were analysed using thematic analysis.The informal teacher communities allowed the tutors to engage in a dialogue with colleagues and share questions, solutions, and interpretations. The teacher communities also provided opportunities to explicate tacit expertise, which helped the tutors to develop an idea of their role and form a frame of reference for their own experiences. Furthermore, the communities enhanced the tutors’ sense of belonging. The tutors felt more secure in their role and they felt valued by the organisation due to the teacher communities.This study shows that informal teacher communities not only support the professional development of tutors, but also validate and strengthen their identity as teachers. They seem to provide a dialogical space where informal intercollegiate learning is stimulated, stories are shared, tacit knowledge is made explicit, concerns are shared, and teacher identity is nurtured.The online version of this article (doi:10.1186/s12909-016-0632-2) contains supplementary material, which is available to authorized users. Many medical teachers have never been trained to teach. This becomes especially problematic when they come to teach in student-centered curricula, which require specific skills, like facilitating active and self-directed learning of students. Medical teachers usually learn these facilitating skills informally from peers in the situated social practices of the everyday workplace. Since the informal learning that occurs from peers is crucial to the professional development of medical teachers, several authors have argued that more research needs to be conducted on peer learning –3. One oEmpirical research on the actual practice of the peer learning of medical teachers, however, paints a different picture than the one described by situated learning theory. In the practice of teaching medicine, learning from colleagues and peers seems to be rather limited; feedback from colleagues is “rare and often unhelpful” , 7. InstSome have argued that the limited opportunities for peer learning are related to the tacit nature of teaching expertise. As teaching expertise is grounded in professional activity rather than in simple rules, it cannot easily be expressed . The tacdomain that members share, with recurring sets of problems. The domain is the cohesive factor in a community, i.e. what brings the members together. The community’s sense of identity is rooted in this shared domain.The community, in the form of the interactions and relations between the members. The continuity and quality of interactions between members determines their willingness to share questions and ideas.The practice is the understanding of the domain that the community members share, including approaches, frameworks, ideas, standards, styles, language, stories, theories, models, principles, and so on. This ranges from tangible objects, like tools and documents, to less concrete displays of competence, such as the ability to recognise a certain problem.The One way to create a dialogical space is to foster teacher communities, i.e. informal groups of teachers who gather voluntarily and regularly in order to develop and share knowledge with and from each other . In his teacher communities in this study.Unfortunately, Wenger uses the phrase “community of practice” for both the natural communities (as described in his 1998 work) and the cultivated, developed communities he described in his later work with McDermott and Snyder in 2002. The use of the same label for two different contexts may lead to confusion. In order to avoid this confusion, we adopted the label Teacher communities may be particularly helpful in fostering the informal peer learning among tutors especially in the early phases of the implementation of undergraduate student-centered or problem-based curricula , 22. NewNo research has yet been reported on teacher communities in the field of medical education. This is surprising, since several authors have ascribed to the potential of teacher communities , 22–24. The existing studies on teacher communities do not give insight into the processes that make informal teacher communities effective. Regehr  recommenThis study investigated the role of informal teacher communities in enhancing the informal peer learning among tutors in an undergraduate student-centered curriculum that was in its early stage of implementation. The research question guiding our study was: “What are the processes that make informal teacher communities effective in supporting the professional development of teachers?”In order to study the processes involved in teacher communities, we employed a qualitative approach. The processes were observed. In order to understand how the participants had experienced the processes, semi-structured interviews were conducted with the tutors.The study took place at a Dutch medical school that offers a 3-year bachelor’s programme and a 3-year master’s programme . Two yeaClinical departments as well as non-clinical departments , were required to provide a certain number of tutors to guide small groups (depending on the size of the department), for which these departments received remuneration. Departments differed in the way they selected tutors; at some departments tutors volunteered, at others they were assigned to the task. Before starting their task, the new tutors attended a 2-day preparatory training course to acquire the necessary skills. In the first day of the training, the student-centered curriculum and the role of the tutor were explained. Through discussions and role plays, the tutors practiced and reflected on the skills required for facilitating self-directed learning in small groups.The role of “tutor” was new to the medical school, although some departments had previously employed small group learning. Not all staff members were enthusiastic about the change of the role of the teacher from expert to facilitating tutor. During the first 2 years of the new curriculum, many tutors struggled with their new role. Many were confused about the type and amount of guidance they should give to the students, and struggled with how to assess students’ professional behaviour. Some also struggled with the perceived low status of the tutor role among their colleagues.Since almost all tutors came from different departments and access to other tutors therefore was not easy, one of the teachers took the initiative to organise teacher communities in which tutors could support and learn from each other. The organising teacher asked the first author (TvL), a professional educator, to be involved. The medical school approved the initiative and supported the teacher communities by providing lunch for all participants and a room for them to meet in . Based on voluntary participation, two communities were formed: one for tutors in year 1 of the bachelor programme and one for the tutors in year 2.shared domain consisted of the shared experience of being a tutor in a recently introduced student-centred curriculum. In the teacher communities, the tutors reflected on shared problems they had experienced and exchanged possible solutions. Each meeting was led by an alternating pair of tutors, who chose a topic and decided how to discuss it. Thus, the topics were directly related to the tutors’ working practice and held a sense of urgency for them. The topics that were chosen concerned the pedagogical part of the tutor role. , while nine declined because of time constraints. Demographic details regarding the participants are presented in Table During the first semester, TvL took detailed observation notes during and immediately after the meetings of the two communities. As the processes we were looking for were unknown beforehand, we did not use a standardised observation scheme. Instead, we tried to include anything that seemed relevant to the research question from the perspective of the theoretical framework. The notes were qualitative in nature. They included: the content of the discussion, i.e. the questions brought in (domain) and the solutions offered (practice) and any side discussions, and the interactions between the members and any important events or incidents (community).After the semester, TvL held individual semi-structured interviews with the participants.TvL is a female educationalist holding an MSc in Psychology and extensive experience in qualitative methods. The participants were aware of the aim of the interview, namely to reflect on the personal outcomes of the teacher communities (both positive and negative) and on the processes within the teacher communities that had contributed to these outcomes. The interview schedule was developed by TvL and MV and is available online as Additional file The interviews lasted for 60–90 min and took place at the university, without anyone else being present apart from the interviewer and the interviewee. Field notes were made during the interview. The interviews themselves were audiotaped and transcribed verbatim. Summaries of the interviews were sent to the participants for member checking.Data analysis was performed from an interpretivist point of view , which fTvL played a double role due to both serving as a co-facilitator in the teacher communities and being involved in the data collection and analysis. She was therefore not a neutral observer, but was involved in the teacher communities herself. We acknowledge that this might have influenced the findings and our account of them. At the same time, we experienced that this double role actually helped us to understand the participants’ responses during the interviews, and it also helped us to recognise and understand the processes involved in the teacher communities during the analysis. To minimise possible conflicts between the two roles, a second researcher was involved in the data analysis, which assisted TvL in creating distance from her role as co-facilitator. Additionally, the research team, consisting of two scientists from the medical education field and three educational scientists, functioned as a critical review board during the data analysis.In line with earlier studies, we found that the informal teacher communities supported the tutors’ learning and helped them to change their practice. This led to enhanced collegiality and a sense of empowerment. We identified three processes within the teacher communities that were involved in these outcomes: developing a frame of reference for the practice of tutoring; creating a sense of belonging; and valuing the domain of tutoring. Each of these processes is described in more detail below.At the start of the semester, many of the tutors felt insecure about their role. The teacher communities helped them to gradually develop an understanding of what the tutor role entailed. Along the way, they also developed a frame of reference to which they could relate and gauge their own tutoring style and performance.In the discussions concerning problems and difficult situations, a range of possible solutions was often exchanged. In general, the exchanged solutions were of a good quality, and if not, the tutors corrected each other by adding other solutions or other insights. In that way, the tutors collectively negotiated what counted as “good” or “valid” behaviour for tutors and what did not. The contributions of the experienced tutors were particularly helpful, since they could point out what solutions they had tried in the past, as well as what solutions had worked and what solutions had not. This helped the tutors to expand their repertoire.I do believe that I can now give shape to the tutor role, that I can play with the group and I can make sure that the group runs well. .The range of solutions offered also illustrated that there are many different ways in which a tutor can influence the learning process of the student group. This helped the participants to develop an understanding of the different ways of enacting the tutor role, while leaving it to individual tutors to choose the way that best fitted their own situation or preference. When exploring the underlying reasons for choosing one solution or another, the discussions often led to a consideration of the role of the tutor in enhancing students’ learning. These collaborative investigations of what the tutor role entailed helped the participants to make sense of the new concept of student-centred learning and their role in it. This increased their confidence:You can mirror yourself, you can compare yourself with others. You realise that what others say, you thought about that yourself. You get a sort of confirmation of your qualities. .In the interviews, the tutors often mentioned that they found it helpful to know each other’s questions and interpretations of situations. When the interpretations and solutions were different from their own, the tutors re-evaluated their “usual” interpretation of the situation and they felt stimulated to try out different ones. This fuelled their creativity in finding solutions and so expanded their tutoring repertoire. When the problems or solutions were similar to their own, the tutors experienced a sense of recognition and emotional support. In the communities, the tutors realised that other tutors were grappling with similar or more difficult problems and that “it wasn’t just me.” The community thus constituted a source of confirmation of their doubts and helped them regain their sense of competence:You don’t feel alone. You have the idea that you’re not alone, but that you are part of a whole. .Most of the tutors were the only one in their department performing the role of tutor and so they felt rather isolated, having no one in their proximity to discuss their questions with. The teacher communities pulled them out of this isolation and provided them with a sense of belonging or a sense of community:What particularly contributed to this sense of belonging was the recognition that other tutors were experiencing similar problems and therefore understood the struggles that the participants saw themselves facing. In the meetings, the tutors also shared their concerns about the way the student-centred concept was implemented by the educational organisation and communicated to them and the students. They all felt that they were not being heard by the organisation, and that they were not being recognised for their expertise as tutors. Finding out that they all experienced these concerns and frustrations created a sense of belonging, and it helped the tutors to look for ways to take individual and collective action.For me, the communities are a sign that the role of tutor is being taken seriously, that you are doing something that is important. .In the interviews, the tutors also mentioned that they viewed the establishment of the teacher communities as a signal that tutorship was taken seriously and considered an important task by the organisation:People look down on tutorship, at least that’s the idea I have. Not every tutor is enthusiastic about it or takes it very seriously. But now that the teacher communities exist, it looks like the medical school does take it very seriously. .Thereby, the teacher communities counterbalanced the opinions of those clinician and scientist colleagues, who, according to the tutors, regarded tutorship as a “weak” job that “does not take much” because it did not seem to require content expertise. In and through the teacher communities, the tutors felt appreciated and valued in their role:all tutors came to the teacher communities; they interpreted their absence as a sign that these tutors did not take their responsibility as seriously as they did themselves. They sometimes found this rather demotivating.Some of the tutors were disappointed that not Earlier research has shown that when attention is paid to the three fundamental elements of communities of practice , informal teacher communities can enhance the professional development of tutors in an undergraduate student-centred curriculum as well as lead to increased collegiality and a sense of empowerment , 26. Thistorytelling, which can be described as the vicarious sharing of personal stories in which teachers describe professionally significant experiences [stories to relate to.The first underlying process is that the informal teacher communities helped the tutors to build a frame of reference for the practice of tutoring. The teacher communities thus helped them to make sense of their role in the student-centred curriculum and in their acceptance of it . Of parteriences , 33. Stoeriences , but alsThe second underlying process we found is that a sense of belonging was created in the informal teacher communities. Several aspects were involved in this. First, the teacher communities allowed the tutors to create connections with tutors from other departments. As in most student-centred or problem-based learning curricula , the tutFinally, we found that the tutor role was valued and considered important in teacher communities. Valuing the role is important for the intrinsic motivation for teaching , 34. In Our study is limited to the role of informal teacher communities in enhancing the professional development of tutors in an undergraduate student-centred curriculum. As we have not investigated teacher communities for other teaching roles, our study has limited generalisability. However, our aim was to explore the processes that make teacher communities effective. We believe an exploratory study of two groups forms a good starting point for achieving this objective, although further research is needed to extend our work to other groups and contexts. Comparison of successful and less successful communities would be a valuable way to fully understand the processes that make teacher communities effective. It would also be worthwhile to explore the value of teacher communities for other roles than tutors in undergraduate student-centred learning  or for clinical teacher roles in postgraduate settings. Positive experiences have been reported in the Dutch context , 37, butIt is also relevant to point out that though the tutors themselves felt more secure in their role, we do not know whether the tutors actually performed better than those who did not participate in the teacher communities. This would also be an interesting question for further research. A further important point to note is that the two teacher communities in our study were active in a particular context, namely in the early stage (i.e. the third year) of a new curriculum. In this particular context, the organisation had not yet fully developed the tutor role at the time of the study.The teacher communities are still active in our medical school, and are now facilitated by some of the active participants of the first groups. The number of participants is smaller than in the first years though. The teacher communities seem to be particularly appealing to novice tutors, who find a sense of belonging there and an opportunity for reflection, and less so for more experienced tutors, who set their priorities differently. Further research is thus needed to identify how long teacher communities can create value for the participants and for the organisation, as well as to determine what type of value is being created . It woulDepending on one’s point of view, the double role of researcher and co-facilitator could be viewed as either a limitation or a strength. We do acknowledge that the double role might have influenced the findings to some extent, but we also view it as a strength because it helped us to recognise and understand the processes involved in the teacher communities. We also believe that it helped us to ask supplementary questions during the interviews. We took measures to minimise the risk that participants would feel reluctant to mention any negative aspects of the teacher communities and to minimise the risk of biased interpretation during the analysis.This study shows that informal teacher communities not only support the professional development of tutors in student-centred curricula, but also validate and strengthen their identity as a teacher. They seem to provide a dialogical space where informal intercollegiate learning is stimulated, stories are shared, tacit knowledge is made explicit, concerns are shared, and the teacher identity is nurtured. A community around the shared practice of being a tutor is hard to find in the medical schools, but we found that it can be deliberately cultivated in the form of teacher communities. Our study showed how a theoretical framework like that of Wenger and colleagues  can infoAt the time of the study, approval from an ethical committee was not required for educational research in the Netherlands, and a national ethical review board for medical education research did not yet exist . ParticiThe interview schedule is available online as an additional file. Data will not be shared since we have not taken permission for this from the participants.


Metarhizium anisopliae against larvae of the dengue vector Aedes aegypti. Our aim was to increase the effectiveness of the fungus for the control of larval mosquito populations.Entomopathogenic fungi are potential candidates for use in integrated vector management and many isolates are compatible with synthetic and natural insecticides. Neem oil was tested separately and in combination with the entomopathogenic fungus M. anisopliae was confirmed using five conidial concentrations (1 × 105 to 1 × 109 conidia mL−1) and survival monitored over 7 days. Two concentrations of fungal conidia were then tested together with neem (0.001 %). Survival curve comparisons were carried out using the Log-rank test and end-point survival rates were compared using one-way ANOVA.Commercially available neem oil was used at concentrations ranging from 0.0001 to 1 %. Larval survival rates were monitored over a 7 day period following exposure to neem. The virulence of the fungus A. aegypti larvae reducing survival to 18 % with S50 of 2 days. Neem had no effect on conidial germination or fungal vegetative growth in vitro. Larval survival rates were reduced to 24 % (S50 = 3 days) when using 1 × 109 conidia mL−1. Using 1 × 108 conidia mL−1, 30 % survival (S50 = 3 days) was observed. We tested a “sub-lethal” neem concentration (0.001 %) together with these concentrations of conidia. For combinations of neem + fungus, the survival rates were significantly lower than the survival rates seen for fungus alone or for neem alone. Using a combination of 1 × 107 conidia mL−1 + neem (0.001 %), the survival rates were 36 %, whereas exposure to the fungus alone resulted in 74 % survival and exposure to neem alone resulted in 78 % survival. When using 1 × 108 conidia mL−1, the survival curves were modified, with a combination of the fungus + neem resulting in 12 % survival, whilst the fungus alone at this concentration also significantly reduced survival rates (28 %).1 % neem was toxic to The use of adjuvants is an important strategy for maintaining/increasing fungal virulence and/or shelf-life. The addition of neem to conidial suspensions improved virulence, significantly reducing larval survival times and percentages.The online version of this article (doi:10.1186/s13071-015-1280-9) contains supplementary material, which is available to authorized users. Aedes aegypti, affecting 50–100 million people annually. An estimated 500,000 people with severe dengue require hospitalization each year . Figure A. aegypti larvae exposed to five concentrations of neem oil. The survival curves were significantly different using the Log-Rank test . The three highest concentrations grouped together, whilst the two lower concentrations were also similar, both causing low mortality rates. A concentration of 0.001 % neem resulted in larval survival rates (77 %) similar to those of the control group . Therefore, this concentration was chosen for use in combination with M. anisopliae and further tested against A. aegypti larvae.A 1 % concentration of neem oil resulted in the lowest larval survival rate (18 %) on the seventh day of evaluation, compared to the other concentrations tested here  on the seventh day of evaluation . The larvae exposed to concentrations 1 × 109 or 1 × 108 conidia ml−1 both displayed S50 values of 3 days. Figure A. aegypti exposed to five concentrations of the fungal isolate used here. The survival curves were significantly different using Log-Rank comparisons . The survival curves for the two highest conidial concentrations were similar, whilst the three other fungal concentrations grouped together resulted in low levels of larval mortality.Survival rates of larvae exposed to different concentrations of conidia were significantly different from each other . The survival rate of larvae treated with neem only (78 %) was statistically similar to that of the controls . The median survival (S50) was 3 days following exposure to fungus + neem.The survival rate of larvae exposed to a combination of neem + fungus was 36 %. This was significantly different to the survival rates of larvae exposed to just the fungus (74 %) or the controls (81 %) [F 8 conidia ml−1) with neem (0.001 %). Table  = 156.04; p <0.01). Larval survival when exposed to neem alone was not significantly different (p <0.01) to the control survival values (90 %). Increasing the concentration of fungus slightly decreased the survival rate of larvae exposed to both fungus and neem when compared to the previous experiment, although it should be observed that the fungus alone at 108 conidia ml−1 also resulted in low levels of survival. All survival curves were significantly different . The S50 value for insects exposed to fungus alone was 3 days whilst a combination of fungus (108 conidia ml−1) and neem resulted in a S50 value of 2 days. A two way analysis of variance was performed to compare the results for larval survival when using the two different concentrations of fungal inoculum. The survival rates when comparing the two conidial concentrations were shown to be significantly different  = 39.2; p <0.01).A second set of experiments was carried out to test the combination of a higher concentration of fungus . Although Integrated Vector Management (IVM) strategies are encouraged by the WHO, they are less often utilized in vector control programmes. This could be one of the reasons why insecticide resistance has quickly developed in vector populations. Research on the possible use of conventional insecticides together with biological control agents (BCAs), or the combination of BCAs with phytochemicals is a new and exciting field.Metarhizium anisopliae against A. aegypti larvae by combining fungal conidia with relatively low concentrations of neem oil. Although neem can be used on its own to control mosquito larvae, this is unlikely to be economically viable for the large geographical areas that would need to be treated. Fungi are considered promising biological control agents for use against mosquito larvae, although any measures that could further reduce kill time in the field are of great interest. We have previously shown that an isolate of M. anisopliae (CG 144) was capable of causing a 90 % larval kill rate under laboratory conditions over a 7 day period [M. anisopliae (ESALQ 818) was also highly virulent to Aedes larvae, with a S50 of 2 days. This isolate was shown to have a half-life of 10 days in tests carried out under laboratory conditions.Here we investigated neem oil as a possible adjuvant for use with entomopathogenic fungi. It was possible to significantly increase the virulence of y period . AnotherM. anisopliae was highly efficient at killing A. aegypti larvae, with 60–90 % mortality observed 72–96 h post-inoculation, however, larval death was not the result of a normal infection process [M. anisopliae is a terrestrial fungus and is therefore not adapted to an aquatic environment.A recent study has shown that an isolate of  process . Butt an process  presenteAedes, Culex and Anopheles [The potential use of phytochemicals for the control of insect vectors has been the subject of many studies (for review see: ). Amongsnopheles , 15, 18.Among the compounds found in neem seed, azadirachtin is the main biologically active component. The presence of azadirachtin in neem seeds has been highly correlated with bioactivity against insects . At a phA. aegypti larvae and have been shown to affect development of immature stages of this insect, resulting in significant reduction in adult emergence [An. stephensi and An. gambiae, the administration of pre-determined doses of neem led to inhibition of feeding and reduction in oviposition as well as significant reduction in the formation of pupae and reduced adult emergence [The lethal effects of neem based products were evaluated against mergence , 23. In mergence .An. stephensi larvae [A. aegypti when 1st instar larvae were continuously exposed to the extracts [The toxicity curves of the commercial neem product used here were grouped together into three concentrations with high toxicity (0.1 % – 1 %) and two concentrations, which displayed low toxicity (0.001 and 0.0001 %). The three highest concentrations resulted in between 18 and 30 % survival, whilst the two lower concentrations resulted in 77–80 % survival. The highest concentration of neem tested here (1 %) resulted in a 50 % reduction in survival rates over 2 days. Other researchers have shown that neem oil caused 95 % mortality when used at a concentration of 5 % against extracts .M. anisopliae were tested against adult An. gambiae and C. quinquefasciatus by spraying this formulation onto the netting of cages into which adult mosquitoes were subsequently released [An. gambiae and 12 % for Culex, which was highly effective when compared to neem alone  and fungus in water . In the study of the effects of neem oil formulations of fungal conidia against larvae of An. gambiae, the same authors recorded modifications in adult emergence following treatments over an 8 day period [The use of a combination of neem oil and released . By day y period . Neem foB. bassiana conidia [B. bassiana, inhibiting conidia vegetative growth and decreasing conidial production and viability [B. bassiana when used at 0.5 % and highly toxic at 1.5 %. However, neem seed and leaf extracts were compatible with this fungus. Another study showed that using neem oil at less than 1 % caused no deleterious effects on the fungus Metarhizium acridum and the combination of neem oil with this fungus accelerated locust mortality and increased efficiency of the biological control agent [Before selecting a suitable adjuvant for application with entomopathogenic fungi, it is necessary to test for possible toxicity to the biological control agent. Although many chemical insecticides are compatible with entomopathogenic fungi, some have been shown to have deleterious effects on these organisms. One of the new generations of insecticides, the neonicotinoid imidacloprid, was shown to have no effect on the germination of  conidia . Howeveriability . Neem oiol agent .7 conidia mL−1 + neem at a final concentration of 0.001 %, the survival rates were 36 % as compared to applications of the fungus alone which resulted in 74 % survival. The results for larvae exposed to this concentration of neem alone resulted in 78 % survival. In the experiments using a higher concentration of fungal conidia (1 × 107 conidia mL−1), the survival curves were different, with this combination of fungus + neem resulting in the lowest survival rates seen here (12 %). At the higher fungal concentration, survival rates were also significantly reduced (28 %) when compared to those using 1 × 107 conidia mL−1 alone. As was observed by other workers, combining two control agents requires extensive research to obtain the desired effect. Our aim here was to verify the concentration of neem oil required to increase larval mortality when combined with the fungus without having a negative effect on the fungus itself. As we show here, all neem oil concentrations tested had no effect on the fungus, however, from an economic view point, using low concentrations of neem is advantageous. The results also show that using higher concentrations of fungal conidia alone significantly reduce survival rates. Even so, combining higher concentrations of conidia with low concentrations of neem also had beneficial effects, low total survival and low S50 values. It should be remembered that these assays were carried out under laboratory conditions and further tests under field conditions should be performed.Here we show that when using a combination of 1 × 10The current approach using a combination of a phytochemical and entomopathogenic fungus has multiple benefits, apart from increasing total mortality and reducing time to kill, neem could also increase fungal persistence and reduce the chances of resistance development which has been shown to occur when using mixtures of control agents .


Bilayer and multilayer graphene were directly identified from areas that exhibited Raman characteristics of turbostratic graphene using high-resolution TEM imaging. Raman maps of the pertinent modes reveal large regions of turbostratic graphene on Ni(111) thin films at a deposition temperature of 1100 °C.Single-layer graphene has demonstrated remarkable electronic properties that are strongly influenced by interfacial bonding and break down for the lowest energy configuration of stacked graphene layers . Multilayer graphene with relative rotations between carbon layers, known as turbostratic graphene, can effectively decouple the electronic states of adjacent layers, preserving properties similar to that of SLG. While the growth of AB Bernal graphene through chemical vapor deposition has been widely reported, we investigate the growth of turbostratic graphene on heteroepitaxial Ni(111) thin films utilizing physical vapor deposition. By varying the carbon deposition temperature between 800 –1100 °C, we report an increase in the graphene quality concomitant with a transition in the size of uniform thickness graphene, ranging from nanocrystallites to thousands of square microns. Combination Raman modes of as-grown graphene within the frequency range of 1650 cm Graphene synthesis originated in the 1960s with graphitization of SiC68SLG is a two-dimensional network of spRelative rotations between graphene layers could provide a novel route to overcome the restrictions interfacial interactions impose on the desirable electronic properties of SLG. Relative rotations between adjacent graphene layers can alleviate π-orbital hybridization, thus, restoring the electronic structure of SLG in a MLG configuration24−13025Turbostratic graphene is comprised of multiple graphene layers with interlayer rotations that alleviate orbital hybridization resulting in carrier mobilities similar to that of SLG. Its relevance has been limited by the difficulties associated with its controlled and reproducible growth, along with its quantitative characterization. Turbostratic graphene has been grown tens of layers thick on SiC and observed through Moiré patterns using STM27283031323334353638Graphene has been grown on a variety of transition metal surfaces such as Co(0001), Cu(111), Pd(111), Pt(111), Ir(111), Ru(0001), Ni(111) and polycrystalline nickel. Single crystal substrates are advantageous growth templates as they increase homogeneity of the grown graphene films through a reduction in nucleation sites414345Of recent interest, particularly for deposition on insulating, magnetic and ferroelectric substratesG) in D/IG ratios  and line-shape of Raman spectral features provide quantitative and qualitative information about the structure and properties of graphene303135545556254049515859Raman spectroscopy probes the electronic and vibrational properties of molecules and crystalline materials through inelastic scattering of photons by phonons and is a powerful tool for graphene characterizationFigure 5a40et al.−1, observed from the 1100 °C(1) spectra. A 2D FWHM of this magnitude is on the order of the 2D FWHM reported for SLG32The normalized Raman spectra, shown in 242532404053et al.et al.−1 and 2300 cm−1 differ in intensity and position for SLG, turbostratic (i.e. incommensurate) and Bernal phase (i.e. commensurate) MLG. Of particular interest are the iTALO−, iTALO+, iTOLA, and LOLA modes that are distinct for turbostratic graphene and become convoluted for AB Bernal stacked graphene layers. The iTALO−, iTALO+, iTOLA and LOLA modes are believed to arise from the combination of in-plane transverse acoustic (iTA) and the longitudinal optic (LO), iTA and longitudinal acoustic (LA) and LO + LA modes. Here, we designate the iTALO− mode as TS1 and the iTOLA/LOLA modes as TS2. Additionally, the iTOTA, R and R’ Raman phonon modes, can also be used to classify interlayer coupling333435Rao 1 and TS2 modes are well defined with distinct intensities while these peaks disperse into the background for AB Bernal BLG and MLG. Further, turbostratic graphene exhibits greater TS1 and TS2 peak intensities than SLG, due to stiffening of the phonon modes and, importantly, their peak positions are blue-shifted301 and TS2 peaks are clearly observed in 1 (circles) and TS2 (triangles) peak positions are displayed for all deposition temperatures in et al.et al.1 and TS2 peak frequencies are blue-shifted for all deposition temperatures and the magnitude of their blue-shift is reduced as deposition temperature is increased (1 peak frequencies shown here closely resemble the reported TS1 peak positions of IBLG (i.e. turbostratic BLG) while the turbostratic TS2 peaks are upshifted from reported IBLG TS2 peak frequencies. This observation is clear when examining the 1100 °C samples; the TS1 peak position is within 2 cm−1 of the reported IBLG values while the TS2 peak position is found at higher frequencies. The increased blue-shift seen in the TS2 peak is consistent across all deposition temperatures and could result from an intrinsic difference between the phonon modes of IBLG and as-grown turbostratic graphene investigated here. Finally, the M band30−1 that is present for Bernal phase BLG and MLG, was not detected in our PVD graphene, further supporting the layer rotations conclusion. The presence of as-grown PVD turbostratic graphene is systematically established with features of the 2D peak, combination Raman modes, in conjunction with high-resolution TEM analysis, our focus proceeds to mapping turbostratic graphene with pertinent Raman signatures.ncreased . Moreove2D), averaged TS peaks (I1+TS2)/2(TS), 2D FWHM and 2D peak frequency, shown in 2D Raman map (I >5000) to right (I <5000). Traditionally, higher 2D intensities would be associated to SLG−1 is observed over the area of interest, indicating this region preserves the electronic structure of SLG263259632D and 2D FWHM Raman maps which are direct evidence of single Lorentzian Raman peaks and turbostratic graphene252D, 2D FWHM and 2D peak position profiles. These observations establish the TS peaks as signatures that can be used to spatially map graphene with incommensurate stacking, despite their limited intensity. The regions that exhibit high I2D, The Raman maps of the integrated intensity of the 2D peak  growth regimes in areas that exhibited characteristic turbostratic graphene Raman signatures. The characteristics of the 2D Raman mode and combination iTALO−/iTALO+ (TS1) and iTOLA/LOLA (TS2) Raman modes were employed to investigate interlayer coupling between as-grown MLG layers. Turbostratic graphene was identified using distinct peak intensities and blue-shifted frequencies of combination Raman modes accompanied by characteristic single Lorentzian 2D peak profiles and direct layer quantification through TEM analysis. Raman maps of turbostratic graphene Raman modes reveal significant coverage of turbostratic graphene on the Ni(111) at high deposition temperatures using PVD. The spatial identification of large-area turbostratic graphene using PVD on nickel thin films provides a unique opportunity to explore the fundamental properties and viability of turbostratic graphene for future applications such as data storage and transmission technologies.Using physical vapor deposition (PVD), large-area turbostratic graphene was grown on heteroepitaxial Ni(111) thin films in a continual process with precise control of the deposition temperature, beam flux, and total amount of carbon deposited. SEM images and Raman maps reveal a substantial increase in graphene regions of constant thickness at higher deposition temperatures. Distinct graphene regions of up to 100 μm in diameter were observed at carbon deposition temperatures of 1100 °C. Correspondingly, low Iet al.−9 Torr from a 99.999% Ni source on single crystalline magnesium oxide (MgO(111)) substrates. Due to the difference in lattice parameters between Ni(111) and MgO(111), 100 nm of nickel was deposited at 300 °C to promote single domain growth and decrease the surface roughness of the thin filmThe growth method present here has been adapted from Iwasaki −1, the G band was designated 1560–1600 cm−1, and the D band was designated 1325–1375 cm−1. The TS1 and TS2 peaks were designated 1860–1910 cm−1 and 2000–2050 cm−1, respectively. The peak intensity ratios, seen in The Raman spectra and Raman maps were taken using a Witec Confocal Raman Microscope Alpha 300R using a 532 nm laser wavelength. The 25 μm × 25 μm Raman maps were constructed from 250 × 250 point spectra. The Raman maps for each deposition temperature were taken of the same region, respectively. For all integrated intensity maps, the 2D band was designated 2670–2730 cmThe SEM images were taken using a JEOL 7600F Analytical SEM at an acceleration voltage of 2 kV and a working distance of 3mm in order to excite secondary electrons from the graphene layers. The TEM images were taken using a double aberration corrected JEOL ARM 200F at an acceleration voltage of 80 kV to mitigate any possible beam damage. The EBSD data was taken at an acceleration voltage of 20 kV and incidence angle of 70°. The mapping of the crystalline orientation takes into account this 20° offset geometry from normal which is why the [111] index is just above the pattern shown in How to cite this article: Garlow, J. A. et al. Large-Area Growth of Turbostratic Graphene on Ni(111) via Physical Vapor Deposition. Sci. Rep.6, 19804; doi: 10.1038/srep19804 (2016).


A graphene-based gas sensor model is also developed. The results from graphene and CNT models are compared with the experimental data. A satisfactory agreement, within the uncertainties of the experiments, is obtained. Graphene-based gas sensor exhibits higher conductivity compared to that of CNT-based counterpart for similar ambient conditions.Carbon, in its variety of allotropes, especially graphene and carbon nanotubes (CNTs), holds great potential for applications in variety of sensors because of dangling π-bonds that can react with chemical elements. In spite of their excellent features, carbon nanotubes (CNTs) and graphene have not been fully exploited in the development of the nanoelectronic industry mainly because of poor understanding of the band structure of these allotropes. A mathematical model is proposed with a clear purpose to acquire an analytical understanding of the field-effect-transistor (FET) based gas detection mechanism. The conductance change in the CNT/graphene channel resulting from the chemical reaction between the gas and channel surface molecules is emphasized. NH There is a rapid growth in the development of sensors both in research and commercial applications. Our daily lives can be noticeably influenced by the development and advancement of miniature and/or portable gas sensors capable of accurately detecting analytes in real-time. Sensors with higher sensitivity and selectivity as well as faster response time are desired. Portability, remote operability and cost effectiveness are some of the features receiving considerable attention because of the ease of their implementation. Rapid advancement in nanoengineering as well as the production of faster and more compact integrated electronic components allow for these goals to be reached –6. NanotCNTs were first discovered by Sumio Iijima in 1991  and have2-bonded carbon atoms is tightly packed in a lattice structure [I–V characteristics of graphene/CNT. Operational amplifiers amplify these signals that can be converted to digital format for digital signal processing.Andre Geim and Konstantin Novoselov  discuss tructure . Due to tructure –21. Due 2 is employed to act as a dielectric layer [2, H2O, NH3, CO, and NO molecules that are physically adsorbed on pristine CNT/graphene [As presented in ic layer –24. Whenic layer ,25–29. Tic layer ,30. Numegraphene –32.3 gas molecules are in the atmosphere around the sensor. Under such conditions, NH3 molecules are adsorbed on the surface of the CNT channel and donate electrons to it. This process causes a quite significant change in the electrical properties of the CNT. These strong adsorption effects stem from the inherent properties of gas molecules and the bonding characteristics between these molecules and the CNT [ the CNT –34. It i the CNT .2 and O2 converts the system to p-type semiconductor while the adsorption of NH3 leads to n-type behavior. Similar to CNTs, these rich adsorption effects are caused by the intrinsic property of the gas molecules and the bonding characteristics between gas molecules and graphene [3 molecule adsorption can greatly enhance the conductance [Gas molecules can modulate the electronic structure of graphene in diverse ways. The adsorption of COgraphene . The resductance ,36.We attempt to model the CNT conductance by considering the energy dispersion relation, and deriving the final model by using the Taylor series expansion near the Fermi points, as follows –38:aC-C = 0.142 nm represents carbon–carbon bond length, d denotes CNT diameter and t = 2.7 eV is the nearest neighbour C–C tight binding overlap energy. For the first band gap energy we can simply write EG = (2aC-C·t/d) = 0.8 eV·nm/d (nm). In addition, since the band structure is parabolic near the k = 0 points, we can write for the energy:where the (±) sign has been included to account for the valence and conductance bands.  is the reduced Planck’s constant, kx represents the longitudinal wave vector component along the length of the tube and m* denotes the effective mass of the CNT effective mass depending on the tube diameter [where diameter –40. The L denotes the channel length. Two major factors contribute to the conductance effect on large channels, enabling it to follow the Ohmic scaling law based on the Landauer formula. The first factor, which is independent of length, is the interface resistance. The second one results from the fact that the relation between the conductance and the width is nonlinear and is dependent upon the number of modes in the conductor. However, these modes are the quantized parameters in the Landauer formula in which both factors are interrelated as demonstrated below [where ed below :h is the Planck’s constant, q denotes the electron charge and T is the transmission probability of an electron injected through the channel approximated as T(E) = 1 in ballistic channels [f(E) is the Fermi–Dirac distribution with df/dE exploding to a delta function near the Fermi energy for degenerate statistics. The conductance can be obtained as follows [where channels . f(E) is follows :x = (E – EG)/kBT and the normalized Fermi energy is given by η = (EF – EG)/kBT. This equation can be numerically solved by applying the partial integration method [where n method –46. The n method . is the Fermi–Dirac integral of the order j. The conductance characteristic demonstrates the performance of NH3 gas sensor based on a CNT nanostructure. It has been revealed that when the CNT gas sensor is exposed to NH3, the conductance changes [ changes . We have changes :Gwog, GwgT and GwgF. The first component Gwog, is the conductance without the presence of gas. GwgT is defined as the conductivity changes in the presence of gas depending on the temperature and the last component, GwgF, is based on different values of gas concentration at a constant temperature [3. EG is dependent on temperature and gas concentration. Consequently, we can write:When the sensor is exposed to the gases in different temperatures, we can define three components for conductance, namely perature . The conEF – EG)/kBT explicitly, we obtainWriting η = ζ(n), and ζ(n) is the Riemann Zeta function. In the degenerate limit (η >> 0), which is the operation regime for the nanometer-scale devices, the expressions for the Fermi–Dirac integral can be obtained from . Accordingly, the Fermi–Dirac integral of order –1/2 can be simplified as [where ified as :VDS), the channel region experiences a flow of electrons. Moreover, the relationship between current and conductance can be replaced by Fermi–Dirac integral of the general conductance model of SWCNT as:Based on the current–voltage characteristic of graphene-based FET devices, the gas sensor performance can be evaluated through Vgs is the gate–source voltage and Vt is the threshold voltage.where G and the transmission probability T of carriers from one electrode to another demonstrated by [The underlying operational principle in MOSFET is based on the electron flow between the source and drain electrodes, which can be controlled by the gate voltage. According to Landauer formula, there is a direct proportionality between conductance rated by :Taylor expansion is used to investigate a parabolic relationship involving energy and wave vector :where β is the quantized wave vector given in . The wavThe conductance on large channel following the Ohmic scaling law based on Landauer formula can then be obtained as:E – EG)/kBT and the normalized Fermi energy is η = (EF – EG)/kBT. The performance of NH3 gas sensor based on graphene nanostructure is demonstrated by its conductance characteristic. It has been shown that the conductance changes when the graphene gas sensor is exposed to NH3 [where x =  and gas concentration control parameter (λ) have been introduced. A comparative analysis between the FET-based models for graphene/CNT sensor structures has been carried out, in which the latter has been validated by an experimental work by Peng et al. [I–V characteristics of the gas sensors are considered for the comparative study under exposure to different NH3 concentrations and temperatures. Finally, the comparison between the I–V characteristics of graphene and CNTs under similar conditions shows that graphene exhibits a higher conductivity than CNTs.Outstanding properties such as high sensitivity as well as remarkable carrier transport features make both graphene and CNTs promising candidates for use in nanosensors. It has been observed that these materials experience a measureable change in conductance levels when exposed to NHg et al. . Aiming


We observe three-color domains with three different ripple directions, which meet at a core. Furthermore, the closed domain is surrounded by an even number of cores connected together by domain boundaries, similar to topological vortex and anti-vortex pairs. In addition, we have found that axisymmetric three-color domains can be induced around nanoparticles underneath the graphene. This fascinating configuration of ripple domains may result from the intrinsic hexagonal symmetry of two-dimensional graphene, which is supported by theoretical simulation using molecular dynamics. Our findings are expected to play a key role in understanding of ripple physics in graphene and other two-dimensional materials.Ripples in graphene are extensively investigated because they ensure the mechanical stability of two-dimensional graphene and affect its electronic properties. They arise from spontaneous symmetry breaking and are usually manifested in the form of domains with long-range order. It is expected that topological defects accompany a material exhibiting long-range order, whose functionality depends on characteristics of domains and topological defects. However, there remains a lack of understanding regarding ripple domains and their topological defects formed on monolayer graphene. Here we explore configuration of ripple domains and their topological defects in exfoliated monolayer graphenes on SiO Two-dimensional crystalline systems and elastic membranes often exhibit spontaneous buckling, bending, rippling, and/or wrinkling. These interesting phenomena arise from the large degree of deformation between bending and stretching caused by external mechanical stimuli, because the elastic energy for stretching or compressing the sheet decreases as a result of out-of-plane deformation122347The surface corrugations are expected to strongly influence the electronic properties of graphene910122/Si substrate can be observed using contact-mode atomic force microscope (AFM)1518192021Previously, it was reported that ripple domains in exfoliated monolayer graphene on SiO2/Si substrates using transverse shear microscope (TSM), which are difficult to be spatially resolved using transmission electron microscope (TEM) or scanning tunneling microscope (STM). We observe three-color domains with three different ripple directions, which meet at a core. Each closed domain is surrounded by an even number of cores connected together by domain boundaries, similar to topological vortex and anti-vortex pairs. In addition, we have found that axisymmetric three-color domains can be induced around nanoparticles underneath the graphene. This fascinating configuration of ripple domains may result from the intrinsic hexagonal symmetry of two-dimensional graphene, which is supported by theoretical simulation using molecular dynamics. Our findings are expected to play a key role in understanding of ripple physics in graphene and other two-dimensional materials.Here, we explore configuration of ripple domains as well as related topological defects and their response to stress in exfoliated monolayer graphenes on SiO2/Si substrate by mechanical exfoliation method and confirmed by Raman spectroscopy  or anti-vortex . The vortex (blue dot) and the anti-vortex (red dot) are always paired. The nearest neighbor of a vortex along a continuous domain boundary is an anti-vortex and vice versa. Moreover, we can observe this regularity of ripple domain network patterns in other regions of our large monolayer graphene .To investigate the response of ripple domains with external stimuli, we carried out high humidity exposure experiments24We now focus on the closed ripple domains that are surrounded by domain boundaries connected to vortices and anti-vortices. It is obvious that there are two γ  and two Vortices that can be considered to be a topological defect are commonly observed in condensed matter, including optical vortex patterns in liquid crystal20181920212226181920282/Si substrate. Biaxial stress on graphene can be expected near graphene–particles contact position. We investigated four different regions around the particles, as indicated by red squares in i.e., two sets of three-color domains , we observed unique configurations of ripple domains in the exfoliated monolayer graphene where the local stress develops around folded or wrinkled edges and external particles. The redistribution of the strain results in an interesting network of three-color ripple domains with three different ripple directions. Seemingly irregular domain networks exhibit an arrangement of three-color domains meeting at one point (core) and the emergence of vortex and anti-vortex pairing as a new type of topological defect in graphene. In addition, an extraneous particle induces a central force on the surface of graphene, leading to radial stress that results in six-ripple domains around it. The rippling is caused by the coupling between the radial zigzag directional tension and the azimuthal armchair directional compression due to the unique features of a graphene lattice, which is supported by theoretical simulation using molecular dynamics. Our observation provides an essential understanding of ripple physics for application of graphene in flexible and nanoscale devices.2 (300 nm)/Si substrate under ambient condition. Using an optical microscope, we observed the exfoliated graphene sheets of various sizes and shapes.Mechanical exfoliation method was used to prepare graphene sheets on the SiOWe obtained AFM topography, LFM, and TSM images simultaneously with the contact mode using two AFMs (SPA-300HV from SEIKO and XE-100 from PSIA) at ambient condition. Because scan size of PSIA XE-100 AFM (50 μm) is much larger than that of SEIKO SPA-300HV AFM (20 μm), PSIA XE-100 AFM has the advantage for observing a large area of the graphene sheet, as shown in 15We performed atomic molecular dynamics on graphene using the consistent valence forcefield (CVFF)J.S.C., T.C. and B.H.P. planned the experiments; Y.P., M.J.L. and J.S.C. prepared samples; Y.P. carried out AFM measurements with the help of J.S.C. and M.J.L.; M.P. and H.L. performed and analyzed the calculations; Y.P., J.S.C., T.C., Q.J. and B.H.P. interpreted the results; all authors participated in discussion and writing the manuscript.Supplementary Information