Graphene-based nanomaterials have gained a lot of interest over the last years in flexible electronics due to their exceptional electrical, mechanical, and optoelectronic properties, as well as their potential of surface modification. Their flexibility and processability make them suitable for electronic devices that require bending, folding, and stretching, which cannot be fulfilled by conventional electronics. These nanomaterials can be assembled with various types of organic materials, including polymers, and biomolecules, to generate a variety of nanocomposites with greater stretchability and healability, higher stiffness, electrical conductivity, and exceptional thermal stability for flexible lighting and display technologies. This article summarizes the main characteristics and synthesis methods of graphene, its oxidized form graphene oxide (GO), and reduced GO derivative, as well as their corresponding polymeric composites, and provides a brief overview about some recent examples of these nanocomposites in flexible electronic applications, including electrodes for solar cells and supercapacitors, electronic textiles, and transistors. It is considered as the starting point of other carbon nanomaterials with different dimensionalities, such as 0D fullerenes, 1D CNTs, and 3D graphite, since these can be assembled based on this unit . Ot. Ot45]. Exfoliated graphene have also been deposited on flexible poly (PEN) substrates, and the resulting nanocomposite was used as an anode in OSCs. This anode showed a high transmittance, a small sheet resistance, an efficiency of 4.2% and a high stiffness that were maintained after 150 bending cycles. Flexible OSCs incorporating a low-pressure CVD graphene with a PEDOT:poly(ethylene glycol) (PEDOT:PEG) block copolymer were developed. They had very good conductivity and transparency and were applied as anode and cathode in conventional and inverted cells , reachin−2, corresponding to a specific capacitance of 550 F g−1 when the entire surface area is used , th, th−2, c−1, which preserved around 90% of the original value after a hundred of cycles [−1.A self-standing 3D PANI/rGO foam was prepared via an in situ polymerization method with the aid of a template, to obtain a specific capacitance of 700 F gf cycles . In addif cycles , which s2SO4–polyvinyl alcohol (PVA) electrolyte was prepared, which was converted into a textile for wearable electronics. Binder free composite electrodes with multilayer graphene and PPy nanowires have also been prepared, which showed a maximum capacitance of 160 F g−1 for the highest scan speed. Novel flexible graphene/PPy nanocomposite films were manufactured using a pulsed electropolymerization technique [−1 was attained for a whole buildup time of only 2 min  was used as green reducing agent to obtain rGO/TiO2 films. Third, annealing was performed at different temperatures to obtain films with diverse titania crystalline phases. Lastly, PPy was deposited onto rGO/TiO2 composites. The TiO2 nanoparticles improve nanocomposite wettability, leading to high capacitance and good cycling stability. Moreover, different TiO phases had different behavior. Anatase had higher capacitance while rutile had better stability.Graphene/PPy composite fibers with diameters ranging from 15 to 80 μm have been developed via wet-spinning approach . The fibechnique . A maximly 2 min , about fr stages : first, Phenolic resin-based laser-induced graphene patterns have also been prepared for application in flexible supercapacitors . This tyWearable electronic devices, like e-textiles, are of great interest for use in multifunctional fabrics, portable electronic devices, and wearable displays . To accoA novel e-textile that can be prepared as yarns or fabrics was prepared with rGO and nylon-6 . They weIn a recent study, the production scale dyeing approach was used to prepare a fabric coated with GO, which was converted into rGO with a sustainable reductor to yield extremely conducting textile electrodes. Then, using the layer-by-layer method, these textiles were subsequently coated with conductive polymers to develop breathable, flexible, and washable electrodes . The effGraphene is highly valuable for flexible electronics but simultaneously needs band-gap opening for digital applications. This nanomaterial can be applied as an electrode in field-effect transistors (FETs), which need high transparency combined with elevated conductivity. It can act as a source and drain, as well as a channel layer in any type of FET structure. The idea is to find a great capacitive, easily printable, tough, and well-matched material for gate dielectric. Polymeric electrolytes can be an optimum selection, though their time-consuming response with frequency impedes attaining a good TFT performance. Taking into account compatibility with graphene, GO can be chosen as an insulator, though the development of high-quality GO film is essential. In this regard, organic field-effect transistors (OFETs) are more attractive as they can easily be fabricated by printing method and exhibit a good on/off ratio.Highly conductive and flexible graphene-based textile composites for OFETs have been recently developed. They were arranged by vacuum filtration and wet-transfer of GO onto flexible PET textiles combined with the addition of AgNPs. In particular, the transistor devices were fabricated with a bottom-gate top-contact structure, as depicted in −1. The transistors presented excellent stability in terms of device performance, which was preserved following a thousand of bending cycles and preserved for 1 month out of the glove box [Analogously, flexible fiber-type FET with graphene/Ag hybrid fibers as highly conductive electrodes were developed via wet-spinning and an adapted wet-drawing process. The fibers were then mixed with AgNPs, which showed elevated electrical conductivity, up to 16,000 S cmlove box .Portable and wearable electronics are becoming extremely popular, since devices make life more secure, healthier, and more relaxed. In particular, flexible electronics have gone through important progresses in recent years, due to the miniaturization of technology and the wireless revolution. Henceforth, novel materials need to be explored. Graphene is regarded as one of the most talented materials for the next-generation of flexible electronic applications, due to its brilliant optical, mechanical, and other characteristics. However, most of the graphene-based devices developed up to date are time-consuming and need multi-stage fabrication routes, which are neither scalable nor suitable for industrial production. Moreover, they typically display poor electrical conductivity, washability, and flexibility. In order to solve these issues, they can be combined with polymers to manufacture nanocomposites with enhanced stretchability, superior mechanical strength, conductivity, and stability. Another approach is the combination with other nanoscale fillers like silver nanowires, that also display optimal properties for flexible electronic applications . However


Introduction: Endothelial cell (EC) proliferation is a fundamental determinant of vascular development and homeostasis, and contributes to cardiovascular disease by increasing vascular permeability to blood-borne lipoproteins. Rodents have been traditionally used to analyse EC proliferation mechanisms in vascular health and disease; however, alternative models such as the zebrafish embryo allow researchers to conduct small scale screening studies in a physiologically relevant vasculature whilst reducing the use of mammals in biomedical research.In vitro models of EC proliferation are valuable but do not fully recapitulate the complexity of thein vivo situation. Several groups have used zebrafish embryos for vascular biology research because they offer the advantages of anin vivo model in terms of complexity but are also genetically manipulable and optically transparent.Methods: Here we investigated whether zebrafish embryos can provide a suitable model for the study of EC proliferation. We explored the use of antibody, DNA labelling, and time-lapse imaging approaches.Results: Antibody and DNA labelling approaches were of limited use in zebrafish due to the low rate of EC proliferation combined with the relatively narrow window of time in which they can label proliferating nuclei. By contrast, time-lapse imaging of fluorescent proteins localised to endothelial nuclei was a sensitive method to quantify EC proliferation in zebrafish embryos.Discussion: We conclude that time-lapse imaging is suitable for analysis of endothelial cell proliferation in zebrafish, and that this method is capable of capturing more instances of EC proliferation than immunostaining or cell labelling alternatives. This approach is relevant to anyone studying endothelial cell proliferation for screening genes or small molecules involved in EC proliferation. It offers greater biological relevance than existingin vitro models such as HUVECs culture, whilst reducing the overall number of animals used for this type of research. Research highlightsScientific benefit(s)•Endothelial cell (EC) proliferation can be visualised at the single cell level in live embryos.3Rs benefit(s)•Zebrafish embryos can be used to partially replace mouse models in early cardiovascular disease research.•This time-lapse zebrafish embryo proliferation assay is a non-terminal refinement ofimmunolabelling and cell labelling approaches, which require tissue fixation.Practical benefit(s)•Only one tank of adult zebrafish are required to produce transgenic embryos for this method (20-40).•Fewer than 30 embryos are required for time-lapse experiments, thus fewer adult pairs are required which reduces breeding stress.•Use of transgenic zebrafish lines allows for proliferating cells to be visualised in real-time without use of embryo fixation, compared to immunolabelling and cell labelling approaches which require embryo fixation.•The zebrafish EC proliferation assay allows simultaneous quantification of proliferation in up to 24 embryos. Assuming four embryos are used as controls, this makes it possible to compare four treatment groups where each group has n = 5 animals. A comparable experiment using 24 mice would require each mouse to be culled, which incurs significant additional cost and effort, and may be difficult to justify, particularly for screening experiments where the potential benefits of the research are unknown.Current applications•Screening for genes involved in EC proliferation in response to blood flow.Potential applications•As a screening assay to identify new drugs/agents that prevent pro-atherogenic changes in endothelial cell proliferation in response to flow.•Screening for genes involved in migration and apoptosis in response to flow by observing cell movement throughout the time-lapse, effects on apoptosis could be screened by counting the number of ECs undergoing apoptosis either by looking for blebbing of EC nuclei, or using a different transgenic.Endothelial cell (EC) proliferation is vital for vascular development and homeostasis, but is also associated with increased vascular permeability which is a key driver of atherosclerosis.In vitro models can be used for high throughput analysis of the genes and pathways that control EC proliferation, but do not model the interaction of different tissues and are therefore reductionist. Conversely, rodents and other mammals have provided physiologically relevant models, such as the ApoE mutant mouse line which is thede facto model of plaque formation in CVD research. Whilst mice are an excellent model for study of genes involved in atherosclerosis , they are unsuitable for screening multiple genes at once as generating mutants is costly, time consuming and may be difficult to justify due to concerns about the effect of mutations on animal welfare and the number of animals that would be required in such a study. Zebrafish embryos survive by oxygen and nutrient diffusion, thus mutations which are embryo lethal in mammals such as abolished development of ECs (cloche) or targeted knock-down of cardiac troponin (sih) are not lethal in zebrafish embryos. These characteristics mean zebrafish embryos are uniquely amenable to study of genes involved in ECs, and EC responses to flow.Like most forms of human disease research, cardiovascular disease (CVD) studies conventionally identify genes for in-depth research by first applying wider screening approaches. The models used in these types of research have several advantages and disadvantages.in vitro models are multiple. One pair of adult zebrafish can produce hundreds of embryos in one day, and with just an incubator and E3 medium these embryos will each develop a complete cardiovascular system in two days,in vitro models, far greater simplicity than comparable three-dimensionalin vitro models, and comparable biological relevance to mammalianin vivo models but with reduced cost and reduced detriment to animal welfare.Whilst zebrafish embryos are not protected prior to 5 days post fertilization (dpf) in the UK, they are still live organisms and therefore present a partial replacement to use of other animals in CVD research. Nonetheless, the advantages of using zebrafish overThe zebrafish embryo is ideal for studying vascular biology, it is small, optically transparent, and allows manipulation of gene expression using morpholino oligonucleotides–in vivo andin vitro models; immunohistochemistry (a technique widely used to study protein expression in mouse endothelium using theen face technique), DNA labelling (used in cell culture and mice) and time-lapse imaging, to determine which method detected the greatest number of proliferating ECs. Indirect approaches for studying proliferation such as cell number counting, viability, and metabolic activity assays were not pursued as their results can be influenced by migration and apoptosis.Zebrafish have been used to model EC apoptosis and migration in response to blood flow.fli1a:nls-mCherry), (fli1a:LifeAct-mClover),fli1:EGFP) lines were used.,fli1a:nls-mCherry expresses a red fluorescent protein linked to a nuclear localisation sequence under the endothelial promoter, resulting in red fluorescence in EC nuclei. Tgfli1a:LifeAct-mClover expresses a f-actin:mClover fusion protein under the endothelial promoter, resulting in visualisation of the EC actin cytoskeleton. Initially, we intended to compare EC proliferation in embryos with and without blood flow, thus for early experiments (whole mount immunostaining) heart contraction was inhibited by silencing troponin T2A by injection oftnnt2a ATG morpholino at 3 ng final dose (sequence 5′-CATGTTTGCTCTGATCTGACACGCA-3′). As EC proliferation was not detected at this experimental stage, flow and no-flow comparison was stopped to minimise our use of embryos and only embryos with blood flow were used for subsequent experiments.Zebrafish care and experimental procedures were carried out under Project Licence 70/8588 issued by the UK Home Office and local ethical committee approval was obtained. Adult zebrafish were kept at a constant temperature of 28 ± 1°C and at pH 7.5 ± 0.5. They were subjected to a light/dark cycle of 14 hours of light and 10 hours of darkness. Adult zebrafish were fed a diet of artemia and dry Zebrafeed™. For mating, pairs of male and female zebrafish were placed into mating tanks and separated by a divider to allow accurate timing of fertilisation. For experiments where this was not required, a box for embryo collection with a mesh insert and marbles on top was placed in the fish tank to harvest progeny. The Nacre lineAdults: For all experiments described, embryos were produced by pair mating eight male and eight female adults of the same transgenic line. This was done to minimize the chance that zero pairs produced embryos, and to minimise confounders by limiting the genetic diversity of the embryos. Adults do not suffer pain or distress from pair mating and were returned to their home tanks immediately after producing embryos, home tanks contain environmental enrichments such as fake jellyfish and fake seaweed. No adults suffered adverse events as a consequence of this work.Tg fli1:EGFP for PCNA, andTg fli1a:nls-mCherry for EdU) then three embryos were randomly selected for imaging. For time-lapse imaging experiments, embryos were screened forTg flia:nls-mCherry andTg fli1a:LifeAct-mClover, then three embryos were randomly selected for imaging. Embryos not selected for imaging were humanely destroyed using bleach.Embryos: No embryos used in this study exceeded 5.2 dpf. For all experiments described, embryo collection stopped after 60 embryos were obtained. Sex of embryos was not determined (as is standard practice in zebrafish research). For PCNA and EdU experiments, embryos were treated then screened for the endothelial marker ((fli1:EGFP) embryos were fixed in 4% paraformaldehyde (PFA) at 30 hpf to allow screening for proliferation in the main trunk vascular beds, then whole mount immunostaining was performed using anti-PCNA antibody  and AlexaFluor-568 mouse anti rabbit secondary antibody . Colocalization of PCNA andfli1-EGFP was used to define proliferating ECs. Embryos not treated with PCNA antibody were imaged during protocol optimization to ensure the specificity of PCNA antibody. Detection of cell proliferation was defined as where a nucleus is both positive for PCNA and forfli1-EGFP.TgThe Click-iT™ EdU Cell Proliferation Kit (Thermo Fisher) was used to label DNA of proliferating cells with AlexaFluor-488. The protocol was modified for zebrafish as follows:fli1a:nls-mCherry) were collected at 30 hpf and incubated in EdU solution  at 28°C for 1 h. Larvae were fixed in 4% PFA overnight then washed and transferred to permeabilization solution (supplied in kit) for 1 h. Permeabilised larvae were then treated according to manufacturer protocol. Proliferating ECs were defined as cells positive forfli1a:nls-mCherry and EdU.Zebrafish larvae with Tg (tnnt2a morphant embryos that lack a heartbeat. The morphant phenotype (no heartbeat) was observed in all (100%) of injected embryos, survival is indistinguishable from wildtype up to the time point tested (30 hpf).Standard light microscopy was used to screen out dead or undeveloped embryos, and to identifyfli1a:nls-mCherry orfli1:EGFP after they were non-terminally anaesthetised using E3 medium. Fluorescence screening was done using a ZEISS Axio Zoom V16 at 28 hpf. Zeiss filter set 63  was used. Camera mode was used and pixel binning set to 5 × 5 to maximise sensitivity to the fluorophore. Screened embryos were washed in E3 media without tricaine, then transferred into fresh E3 prior to further imaging.Fluorescent microscopy was used to identify and select embryos containingFor confocal imaging, embryos were non-terminally anaesthetised using tricaine and mounted in 1–2% agarose over which standard E3 medium was placed. Still images were taken using a Nikon A1 confocal microscope at 1024 × 1024 resolution (0.62 μm per pixel), z-stacks were taken in 8-μm increments. Time lapses were taken using a Zeiss LSM880 in Airyscan mode. Images were captured at 1848 × 1848 resolution (0.47 μm per pixel), z-stacks were taken in 3.43-μm increments. Images were analysed using NIS Elements or ZEN respectively. Composite images were generated by performing maximum intensity projection of the stack data, no other processing was performed. For both microscopes, excitation was done by a 488 nm and 568 nm laser.,fli1a:nls-mCherry nuclei visibly divide over the course of the time lapse. Experimental embryos were then euthanised using bleach.For light sheet microscopy,The number of proliferation events detected using EdU labelling, time-lapse confocal microscopy and PCNA immunolabelling were compared using a two-way ANOVA (n = 3 embryos per each group). Each animal was treated as a single unit of analysis. Analysis was done using GraphPad PRISM (RRID:SCR_002798), an open-source alternative to this is JASP (RRID:SCR_015823).4 and 0.33 mM CaCl2 in dH2O. 4% PFA: paraformaldehyde diluted to 4% with E3 (One month shelf life when stored at 4°C).tnnt2a MO: diluted to a 100 μM stock with dH2O. Stock concentration should be determined using a Nanodrop and diluted to 3 ng/nL for microinjection. PBS: phosphate-buffered saline (ThermoFisher) diluted from 10× stock. PBST: phosphate-buffered saline with Tween-20. PBS made to 0.1% Tween-20. PDT: phosphate-buffered saline with 1% DMSO and 0.1% Triton X-100. TRIS buffer: tris base diluted in dH2O to 1 M stock. 150 mM solution was prepared from stock and pH adjusted to 8.0. Tricaine: 4 g tricaine methanesulfonate in 1 L dH2O pH 7–7.5. Blocking solution: 1% bovine serum albumin in PBS. Goat serum: goat serum stock (frozen) diluted to 10% in blocking solution. Bovine serum albumin (BSA): stock albumin powder should be added to dH20 to make 10% (w/v) BSA solution, this solution should be aliquoted into 1.5-mL Eppendorf tubes and frozen for use as needed. Click-IT EdU AlexaFluor-488 Flow Cytometry Assay Kit – ThermoFisher C10420.E3 medium: 5 mM NaCl, 0.17 mM KCL, 0.33 mM MgSO1)Collect embryos and transfer to a petri dish of E3 media.2)Incubate embryos at 28°C until at least 24 hpf.3)Insert forceps to gently pierce the embryo chorion (Sup. 1 – 1).4)Gently open forceps to tear the chorion (Sup. 1 – 2).5)Open forceps until embryo is free of the chorion (Sup. 1 – 3).1)fli1-EGFP embryos and transfer to a petri dish of E3 media then incubate at 28°C to 30 hpf.Collect2)Dechorionate embryos.3)Fix in 4% PFA overnight at 4°C – now protect samples from light as far as possible until imaging.4)Wash in room temperature PBST  on a rocker for three 10-min washes.5)Wash in room temperature TRIS buffer (150 mM pH 8.0) on a rocker for 5 min.6)Equilibrate embryos in TRIS buffer at 70°C for 15 min.7)Wash in room temperature PBST on a rocker for two 5-min washes.8)Rinse with distilled water on ice.9)Wash in chilled acetone (−20°C) for 15 min.10)Rinse with distilled water for three 5-min washes.11)Block with goat serum (10%), BSA (2%), and PBT  for 4 h at 4°C.12)Incubate with primary antibody overnight at 4°C (Dilute PCNA 1:250 in blocking solution).13)Wash in PBT for four 30-min washes.14)Incubate with secondary antibody (AF-568 anti-rabbit diluted 1:500 in blocking solution).15)Wash in room temperature PBST five times for 5 min.16)Mount as described in ‘Mounting individual zebrafish embryos for confocal imaging’.17)Image using a confocal microscope as described above.1)fli1a:nls-mCherry embryos and incubate at 28°C until 30 hpf.Collect Tg2)Dechorionate embryos.3)Incubate embryos in labelling solution  at 28°C for 1 h.4)Fix in 4% PFA at 4°C overnight.5)Wash with PBST for three 5-min washes, rock samples each time.6)Permeabilise in 1× saponin solution  for 1 h at room temperature while rocking samples.7)Incubate with reaction cocktail  for 1 h in the dark at room temperature whilst rocking.8)Wash five times in PBST for 5 min whilst rocking.9)Mount as described in ‘Mounting individual zebrafish embryos for confocal imaging’.10)Image using a confocal microscope as described above.1)Take treated embryos and mount on glass slides in vectashield (RRID:AB_2336789). Embryos must be laid flat on their side. This occurs naturally for young embryos (~30 hpf). If older embryos are used, the yolk may prevent embryos from lying flat, in this case the embryo yolk and head can be cut off using a scalpel.1)fli1a:nls-mCherry embryos and incubate at 28°C until 26 hpf.Collect Tg2)Dechorionate embryos using forceps.3A)For live imaging using LSM, suspend embryo(s) in melted low-melt agarose (37°C 0.8–1.2%), then use a plunger to draw the embryo(s) into a glass capillary. Wait 2 min for the agarose to set, then suspend the embryos (within the agarose column) into the LSM by gently pushing the plunger.3B)For confocal imaging, add embryo(s) to melted low melt agarose (37°C 0.8–1.2%) then draw up embryo(s) and decant into an Ibidi μ-Dish 35mm (Ibidi cat no – 81156). Use thin-tip forceps to gently push the embryo into a side-mounted position, then wait for agarose to set.4)Image using appropriate microscope ensuring 587 nm excitation is active. Adjust magnification to visualise as much of the trunk as possible whilst retaining nuclear resolution . Z-range should traverse the entire trunk vasculature and Z-interval should be set no lower than 8 μm.5)For euthanasia, discard embryos into bleach. For revival, return embryos to E3.1)Collect embryos and transfer to 28°C E3 media.2)Take a glass slide and place it in a petri dish so it is unable to move further to one side.3)With a Pasteur pipette, draw up embryos then, holding the glass slide petri dish slightly tilted, dispense embryos along the edge of the glass slide (Sup. 2).4)Prepare a glass needle  using a Sutter P1000 glass capillary puller. Suitable settings are heat 475, pull 60, vel 80, time 135, pressure 500. Refer to the user manual for capillary pulling optimization.5)tnnt2a morpholino to a glass needle and calibrate the ejection volume using a graticule .Add injection media containing 3 ng/nL of6)Using a manipulator (World Precision Instruments M3301) and PicoPump (H. Saur PV820), inject 1 nL of morpholino into each embryo until desired number of treated embryos are obtained (some extra embryos should be treated to account for failure to develop and death).We analysed EC proliferation in the trunk vasculature of zebrafish embryos because this vascular bed allows simultaneous imaging of the intersegmental vessels and dorsal aorta . We atteTg fli1a:nls-EGFP) were fixed at 30 hpf then treated with anti-PCNA antibody. Imaging of the embryo trunk detected many PCNA+ cells in the caudal vein plexus (CVP) and none in the ISVs , whereas immunostaining is restricted to detecting markers of proliferation at a single time point. To assess the effectiveness of EdU staining, transgenic embryos expressing endothelial nuclear localised mCherry (Tgfli1a:nls-mCherry) were treated with EdU from 30 to 31 hpf. Embryos were then fixed and treated with fluorescent azide. Between zero and three proliferating ECs were detected in the trunk of treated embryos, with detection occurring in the DA, but not the ISVs (The cell labelling approach works by incorporation of thymidine derivatives such as EdU into newly synthesised DNA. The modified thymidine is labelled with a fluorescent azide thereby enabling the identification of nuclei which have undergone proliferation.he ISVs .fli1a:nls-mCherry) and cytoplasmic LifeAct-mClover (fli1a:LifeAct-mClover) using a light sheet microscope. Using this approach, EC proliferation was quantified by counting the division of EC nuclei in all visible ISVs from 26 to 30 hpf  which has well described toxic effects above the 2% level. 30 hpf .Detection of EC proliferation in the intersegmental vessels was compared for each method. The ISVs are isolated from other tissues, so it was easiest to spot proliferation in this vascular bed. There are also relatively few nuclei in the ISVs, and ISV nuclei are less densely packed than nuclei in the DA/CVP. PCNA did not detect any proliferation events in the ISVs, EdU labelling detected fewer than one proliferation event on average, whereas time-lapse imaging detected an average of nine proliferation events . Two-wayper se, as evidenced by detection of PCNA positive nuclei . This observation is comparable to those observedfli1a:nls-mCherry, fli1a:lifeAct-mClover) enabled direct visualisation of EC nuclei division in proliferation  because of a larger detection window  and7.•Greater breadth of data collected. Time-lapse data could also be analysed for cell migration and apoptosis as desired.Despite this, EdU labelling is unsuitable to analyse the effects of interventions on proliferating ECs because of the limited window of detection and low number of proliferating cells detected. Therefore, we sought a method to capture greater numbers of proliferating EC from zebrafish embryos. We found that light sheet time-lapse microscopy of transgenic embryos  allow real-time imaging of EC proliferation, but lack biological relevance. Whereas establishedin vivo models  are biologically relevant yet cannot be used for real-timein situ monitoring of EC proliferation, and must be culled to isolate vascular tissue for analysis. By using the zebrafish embryo to study EC proliferation, researchers can achieve real-timein situ monitoring of EC proliferation in a biologically relevant model whilst replacing mammals and reducing or eliminating the use of terminal procedures, thus achieving partial replacement and refinement of use of animals in CVD research.This study demonstrates the utility of time-lapse imaging zebrafish embryos to measure EC proliferation. ComparableThis time-lapse approach is more suitable than other methods for quantifying EC proliferation in zebrafish larvae and has applications for studying the mechanisms of proliferation and for screening to detect compounds that modify this process, we are currently using this approach to use zebrafish larvae to screen for genes involved in EC proliferation in response to blood flow. Whilst the manual counting approach used currently is low-throughput, future work could automate detection of proliferation using image macros to enable widespread use of this system for identifying genes involved in atherosclerosishttps://www.doi.org/10.17605/OSF.IO/NV9XE.OSF: Underlying data for ‘Quantifying endothelial cell proliferation in the zebrafish embryo’.The project contains the following underlying data:Raw images of three embryos imaged using the PCNA method, three embryos imaged using the EdU method, and three embryos imaged using the timelapse method.https://www.doi.org/10.17605/OSF.IO/Y2SCW.OSF: Extended data for ‘Quantifying endothelial cell proliferation in the zebrafish embryo’.The project contains the following extended data:Supplementary Figure 1: Dechorionation of zebrafish embryos. 1) Insert forceps into embryo chorion, be careful not to damage the embryo. 2) Gently open forceps to tear the chorion further. 3) Widen the forceps until the embryo has escaped the chorion.Supplementary Figure 2: Preparing zebrafish embryos for microinjection. 1) Place a glass slide in a petri dish and tilt as shown. 2) Draw up embryos in a Pasteur pipette and decant onto the edge of the glass slide as shown. 3) Use the Pasteur pipette to draw up any excess liquid from the petri dish, leaving a row of zebrafish embryos against the glass slide.https://www.doi.org/10.17605/OSF.IO/Y2SCW.OSF: ARRIVE 2.0 checklist for ‘Quantifying endothelial cell proliferation in the zebrafish embryo’.Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the The Article by Bowley et al. describes a novel approach to quantify the relatively slow rates of endothelial proliferation in vivo, using zebrafish embryos, rather than in vitro models. The approach has several advantages over using other animal models to examine endothelial proliferation in vivo, including the potential use of zebrafish lines that use fluorescent reports for cell-type differentiation, the ability to examine proliferation pre-post heart beat, to determine any effects of flow and easy genetic manipulation through genome editing or knockdown with morpholinos. As such, this model provides a flexible platform to examine drug or gene regulation of  proliferation. The text and methodology is clearly described and in sufficient detail to allow other scientists to replicate the experiments described here. As such it is a useful step forward to quantify in vivo proliferation while minimising the use of protected species. The platform is sufficiently flexible to be used to advance the field on this topic.Is the rationale for developing the new method (or application) clearly explained?YesAre the 3Rs implications of the work described accurately?YesIs the description of the method technically sound?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre the conclusions about the method and its performance adequately supported by the findings presented in the article?YesIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?YesAre a suitable application and appropriate end-users identified?YesAre sufficient details provided to allow replication of the method development and its use by others?YesReviewer Expertise:Endothelial biomechanics and endothelial dysfunctionI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al. determine the feasible method to quantify endothelial cell (EC) proliferation which regulates vascular formation and homeostasis in zebrafish embryos to investigate development via a comparison between three different comparative analyses: antibody, DNA labeling, and time-lapse imaging. Although rodent models are predominantly used to explore the mechanism of EC proliferation in cardiovascular disease or development, the authors pointed out there is a limitation - time-consuming, costly, and detrimental to animal welfare. The authors concluded that time-lapse imaging is accurate, precise, scalable, simple, and effective than the other two methods: antibody (PNCA) or DNA labeling (EdU) in EC proliferation study in the zebrafish embryo model. This method paper is necessary to approach EC proliferation in zebrafish and is well designed to compare three different methods, however, there are major and minor points that need to be improved.The authors are focusing on the quantification of EC proliferation using three different methods, however, vascular endothelial growth factor (VEGF) is the most common marker for EC proliferation. Please address why the authors chose PCNA for detecting proliferating cell nuclear antigens and EdU for measuring DNA proliferation instead of VEGF, and it is necessary to be discussed.EdU labeling is a well-established method to quantify S-phase cells. The authors performed EdU labeling in zebrafish embryos for 1 hour and showed that it did not label as many cells as they observed via tracking nuclear division over 6 hours . This does not translate to EdU labelling being unable to provide accurate measurements. Instead, it simply prompts a longer period of EdU incubation. The authors argue that the high concentration of DMSO used in the labeling solution is toxic to embryos for long-term incubation, however, there is no necessity of using DMSO during EdU incubation as EdU is soluble in aqueous buffers. The authors should perform extra experiments on this.On the other hand, there are large amounts of EdU+ cells (green) colocalization with ECs (red) in Figure 3. The authors should also test their hypothesis at different regions of the vascular network, not just ISV.In Figure 2, to see the clear effects of tnnt2 morphant (inhibition of heart contraction) in EC proliferation, the control group (control morphant) should be presented. The authors also need to provide negative control images with only secondary antibodies to validate the staining, as virtually every cell in the image was red, meaning PCNA+. However, PCNA staining should give fewer labeled cells than EdU staining and time-lapse tracking, according to the authors. It can also be that the PCNA antibody is not working well. The authors need to address this.In addition, to rely on the comparison, Figures 2 and 3 should be offered the same enlargement.In Figure 5, two-way ANOVA is an advisable statistical method among the groups.The graphical abstract  is arduous to comprehend because of a lack of a rationale for the limitation of immunostaining and EdU labeling. The authors should offer the reference or evidence to elaborate the experimental design.In this method article, BowleyIs the rationale for developing the new method (or application) clearly explained?YesAre the 3Rs implications of the work described accurately?YesIs the description of the method technically sound?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre the conclusions about the method and its performance adequately supported by the findings presented in the article?YesIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?PartlyAre a suitable application and appropriate end-users identified?YesAre sufficient details provided to allow replication of the method development and its use by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The authors’ clearly set out the context for their study; developing a non-invasive experimental system to accurately capture spatial and temporal information on EC proliferation in the vasculature would be important step forward. Three techniques are explored for achieving these aims. Two  do not appear to work for the intended purpose but it is not immediately clear from the data shown why. More ‘negative’ data would be very helpful in setting out the limitations of these techniques so that others can appreciate why these approaches ‘failed’. One technique, time-lapse imaging of nuclear division (where labelling driven by a EC-specific promoter) does appear to have some future applicability, although the technique, at present, is limited to manual counting and and very low numbers of experiments are reported in support of this approach. There are a number of issues with the current version of the manuscript that require attention:Abstract The abstract refers specifically to n=24 experimental units, where in the example given, n=4 comprise controls and then four groups of n=5 ‘treated’ groups. These numbers seem rather arbitrary and are not referred to in the paper.Introduction / General It would be helpful to include more detail on how the ZF embryo CV system recapitulates key features of mammalian circulatory system. Please provide clarity (and perhaps illustrative detail) on how ZFe’s “complete cardiovascular system” has a “simplicity comparable with that of two dimensional models in vitro” and “comparable relevance to mammalian in vivo models”. This might help uptake by those researchers typically using mammalian models of vascular plasticity. Blebbing of nuclei is not a unique marker of apoptosis. Autophagy and other activation of modes of cellular degradation are characterised by blebbing. The authors imply that apoptosis is the sole mechanism of cell death in ZF embryos. Is this correct? No data on cell death is reported in the manuscript so the references to apoptosis don’t seem to have much relevance in the context of the paper.MethodsHow are male and female zebrafish distinguished?Does the Nacre line have identical vascularization as wild-type?You state that “both transgenic lines use Nacre as background” but the study uses three TG lines.How leaky, or not, is the EC-specific promoter. Can you be sure that expression is only in EC lineages? This is a key issue since your method relies on typing ‘EC-specific’ expression.A reference to the inhibition of contractility by tnnt2a morpholino should be given.It is presumed that that TG-EGFP was used for PCNA and TG-mCherry was used for EdU to avoid the spectral overlap of fluorophores in each instance? This should be stated.More details on how the commercial EdU labelling kit results in the generation of a detectable 488nm signal should be included. What is the concentration of saponin? What is the range of EdU signal developed (i.e. time-dependent accumulation)? How homogeneous is this across cells/FACS populations?Brightfield microscopy is included as the means for discriminating dead or undeveloped embryos are identified. How? No brightfield imaging is included in the paper.Please define Light Sheet microscopy as LSM  laser scanning microscopy.The statement that “587nm excitation is active”. Please include the excitation source and what is meant by “active”.The methods include non-terminal anaesthesia using E3 medium (“Fluorescent microscopy”) or E3 medium and tricaine (“Light-sheet microscopy”). Please clarify. What is the final concentration of tricaine used in anaesthesia?Statistics. Please check that two-way ANOVA is an appropriate statistical test for data where n=3 .degrees C?).Microinjection - it is appreciated that the settings are specific for the Sutter puller but it would be helpful to include units as applicable  but its not immediately clear what itFigure 3 should be presented at the same magnification/zoom as Figure 2. Figure 3 should also be better integrated with your conclusion that EdU labelling was ruled out of contention as a technique for quantifying EC proliferation. Do you have any images showing  the effect of 15% DMSO on sample morphology beyond 1 hour? It is also somewhat misleading to state, as you do in Figure 7, that EdU labelling is limited by low detection of EC proliferation. The problem seems to be that the reagent is toxic which prevents longer-term imaging. It needs to made more clear that EdU is of limited use for mapping cellular proliferation occurring on a time-scale incompatible with reagent toxicity.Figure 4. Your findings suggest that to capture sufficient EC proliferation, an experimental window of up to 12 hours is required. Figure 4 only shows a plus-15min window. Some idea of how time-lapse capture of EC proliferation changes with time would be very helpful. Also, the arrowheads appear squashed and point away from the area you mean to depict.Figure 5 shows the number of proliferation events but this is not that useful. It would be much better if you showed this the number of as the proportion or percentage of mCherry cells scrutinized. ‘n’ is very low here and statistical analysis is not convincing .DiscussionIt is not possible to conclude, from the data shown, that nuclei-imaging is “accurate” . This will need much more data and deeper modes of analysis.As you note, “computer assisted quantification” would augment your approach, but this phrase is vague. What would you want to focus on, and what would need to be taken into account / eliminated etc. More discussion on how you see this approach being potentially worked-up into a medium- to high-throughput screening platform would be helpful.OtherZebrafish welfare and use section. The second-to-last sentence in the first paragraph needs splitting into two sentences. It would be helpful to define what constitutes an “adverse event” here.PCNA and EDU should be described in full at first mention. Has PCNA ever been used as a marker of proliferation during zebrafish CV development? (You state kidney but what about CV…….)The phrase “humanely destroyed using bleach” is inappropriate. It might be better worded as “embryos were destroyed using bleach”. Ditto “euthanised” on pages 5 and 8.The Supplemental Figures do not add to the article and should be omitted. Videos of the actual procedures would be much better.Is the rationale for developing the new method (or application) clearly explained?YesAre the 3Rs implications of the work described accurately?YesIs the description of the method technically sound?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?NoAre the conclusions about the method and its performance adequately supported by the findings presented in the article?PartlyIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?PartlyAre a suitable application and appropriate end-users identified?YesAre sufficient details provided to allow replication of the method development and its use by others?PartlyReviewer Expertise:Network behaviour of cardiovascular cell types / cell imaging / imaging technologies / signal decoding. Referee suggested by the NC3Rs for their scientific expertise and experience in assessing 3Rs impact.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


Such results indicate graphene is an exceptional nanomaterial for future MIR optical interconnects.The mid-infrared  waveband is of great interest for integrated photonics in many applications such as on-chip spectroscopic chemical sensing, and optical communication. Thermo-optic switches are essential to large-scale integrated photonic circuits at MIR wavebands. However, current technologies require a thick cladding layer, high driving voltages or may introduce high losses in MIR wavelengths, limiting the performance. This paper has demonstrated thermo-optic (TO) switches operating at 2 μm by integrating graphene onto silicon-on-insulator (SOI) structures. The remarkable thermal and optical properties of graphene make it an excellent heater material platform. The lower loss of graphene at MIR wavelength can reduce the required cladding thickness for the thermo-optics phase shifter from micrometers to tens of nanometers, resulting in a lower driving voltage and power consumption. The modulation efficiency of the microring resonator (MRR) switch was 0.11 nm/mW. The power consumption for 8-dB extinction ratio was 5.18 mW (0.8 V modulation voltage), and the rise/fall time was 3.72/3.96 μs. Furthermore, we demonstrated a 2 × 2 Mach-Zehnder interferometer (MZI) TO switch with a high extinction ratio of more than 27 dB and a switching rise/fall time of 4.92/4.97 μs. A comprehensive analysis of the device performance affected by the device structure and the graphene Fermi level was also performed. The theoretical figure of merit (2.644 mW Mid-infrared  is a prAmong the on-chip photonic components, optical switches  (electroπP) of 0.39 mW. However, the low thermal conductivity of polymer leads to a long response time of 92.4 μs, and polymers also become opaque in the mid-infrared waveband. The figure-of-merit (FOM) of a TO device combines π-phase-shift power consumption and response time τ in the form of 1/(π·τP), which could be used to evaluate the performance of devices based on different materials and devices platforms [πP of 9 mW, respectively [Two-dimensional materials ,42 own rlatforms . Recentllatforms ,53. To flatforms  or photolatforms ,56. Siliectively . Taking ectively . Graphenectively . ChalcogπP power consumption of 14.42 mW, and comparable FOM of 0.0175 mW−1μs−1 to previously reported silicon TO switches with graphene heaters. Our MZI device showed a high modulation depth of 27.8 dB. Lastly, a complete numerical analysis of the device’s performance was demonstrated. The design strategy of the graphene heater and the performance limits was given. Optimal structural parameters and graphene with a Fermi-level of 0.43 eV for the MRR-based TO switch could achieve an ultrahigh FOM of 2.644 mW−1μs−1. In this context, we demonstrated the first silicon TO switch with graphene heaters operating at 2-μm mid-infrared waveband, which is also considered a new communication window for the next-generation optical communication  to meet 2 box layer by multi-project-wafer (MPW) involved processes. First, the waveguide structures were patterned by deep ultra-violet photolithography and etched by inductive coupling plasma (ICP) process to a ridge depth of 150 nm and width of 600 nm. Next, a layer of 1-µm-thick SiO2 cladding was deposited by plasma-enhanced chemical vapor deposition (PECVD). Then the silica cladding was polished by chemical mechanical polishing (CMP) to a thickness of about 300 nm (150 nm higher than the waveguides). The titanium/gold  contact electrodes were patterned by electron beam lithography (EBL) and deposited using electron beam evaporation. The contact distances were 2.6/7 μm for the MRR/MZI-based switches, preventing the excessive absorption from metal contacts. Then the chemical-vapor-deposition (CVD) graphene [The schematic illustration of the MZI device is shown in graphene  was trangraphene . The loaThe measurement setup is shown in The MRR-type switches play an essential role in silicon photonics. The MRR can behave as a spectral filter, which can be used for applications such as wavelength division multiplexing and sensing. The position and the shape of the dips are very sensitive to structural parameters. In most cases, the spectrum of the fabricated device could not be predicted precisely because of the fabrication error. Therefore, wavelength tuning is necessary for wavelength-relevant applications, and thermally modulating the refractive index of the waveguide in the microring resonator is a straightforward and effective approach. The switching function of an MRR works in the same principle .We fabricated MRR-based switches with a ring radius of 40 μm and a graphene length of 100 μm. The microscope image of the MRR-based TO switch is displayed in η of the switch was calculated to be 0.11 nm/mW by fitting the curve in πP of 14.42 mW, where πP of a cavity-type switch is defined as the power consumption to detune the resonator by a phase shift with a product of π and the full width at half maximum (FWHM) of the resonance peak [3 and the FWHM was 0.505 nm. By decreasing the propagation loss from the waveguide (1.62 dB/cm) [πP power consumption can be further suppressed. This could be improved by optimizing the fabrication process to reduce the scattering loss of the waveguide and doping graphene to ensure less absorption. The sheet resistance of the heater is calculated to be about 125 Ω from the current-voltage (I-V) curve shown in FOM of 0.0175 mW−1μs−1, which is the highest performance among the reported graphene-assisted silicon TO devices operating at the 2 μm waveband.The resonance wavelengths of measured transmission spectra were red-shifted, as depicted in nce peak . It shou2 dB/cm)  and absoThe MZI-type 2 × 2 optical switch is also one of the indispensable components in photonic integrated circuits, which is conventionally constructed by two 3-dB splitters/couplers using multimode interference (MMIs) and connecting waveguides as the two MZI arms. By tuning the optical phase difference of the light between two arms of the MZI, the optical intensity distribution of the output ports can be modulated, realizing optical switching for applications including cross-connection, optical routing, and information processing.We fabricated balanced 2 × 2 MZI TO switches with 100-μm-long graphene on both arms of the MZI to balance the loss difference between the two arms. The microscope view of the switch is shown in the inset of πP of 57.75 mW. Such high-power consumption was due to an extensively long contact distance which will be explained later.The light from two arms of the MZI with variable phase delay was combined by a 2 × 2 MMI, resulting in different power distribution at the output ports. FOM of the switch is calculated to be 0.003 mW−1μs−1.The dynamic response of the switch was also characterized and demonstrated in FOM comparable with TO switches using doped-silicon heaters. Although the doped-silicon TO modulators show better performance at present, our theoretical prediction implies that graphene is a more appropriate heater material at MIR waveband, which will be explained in the following session.To investigate the theoretical performance limits of graphene-based TO-switches, optical modeling of the waveguide modes and finite element method thermal simulations of the graphene phase shifter were carried out using Lumerical MODE solution and the COMSOL Multiphysics package, respectively. 0λ is the free-space wavelength, Δeffn represents the variation of effective refractive index, and gn is the modal group index. Based on the perturbation theory, the effective refractive index tuning ∆effn should be given by a surface integral about temperature, electric field, and refractive index [dn/dT is the material thermo-optic coefficient. The TO coefficients are approximately 1.76 × 10−4 K−1 for silicon [−5 K−1 for silicon dioxide [The thermally induced refractive index change causes the wavelength tuning of TO switches. The wavelength shift corresponding to the refractive index could be expressed by :(1)Δλ=λ0ve index :(2)Δneff silicon  and 1.3  dioxide  at 2 μm,FOM benefiting from the resonance effect. The critical factors for the graphene-on-SOI phase shifter are the cladding thickness (clh), contact distance (cd), and the resistance of the heating region. The measured resistance of the graphene-based MRR resonators is 125 Ω. It contained resistance (gR) from the graphene heater and contact resistance (Rc) between metal and graphene. Considering the sheet resistance of graphene is 1307 Ω/sq, cR would be 4550 Ω·μm. Thus, the divided voltage dU applied on the graphene heater in the simulation is equal to gU∙Rt + R/(Rc), where U is applied voltage across the electrodes. For a given length of graphene heater, the contact resistance would keep the same while distance between the two electrodes varied. clh) and contact distance (cd) on the temperature of the waveguide core. Although the increase of the contact distance cd would entail an increased divided voltage dU on the graphene heater, the increase of total resistance and thermal mass would lead to a decrease in temperature change ΔwgT in the waveguide with the increasing contact distance, shown in clh becomes thicker, the graphene heater will keep further away from the waveguide core. The ΔwgT will also decrease. For an MRR type graphene-based TO switch with a radius r of 40 μm and a heater length hl of 100 μm, the overall wavelength shifting could be expressed as follows:In this section, we focus on the analysis of MRR type TO switch since it could gain much higher λ profile, which is illustrated in η of switches with variable design parameters could be calculated byP represents the change of electric power. The electric power can be calculated by 2/RtU, where tR is equal to g + RcR.Based on Equations (2) and (3), we can convert the thermal profile to the wavelength shift Δclh and cd are presented in clh and cd are also desired for faster operation. The simulated response time is 3.735 μs which is very close to the experimental results of 3.96 μs.The response time of the thermo-optic switch is significant for high-speed light intensity modulation and optical routing. Finite element method (FEM) simulation could also be applied to study the dynamic heating progress. The temporal evolution of the temperature of the waveguide core was simulated under a square-wave driving bias with a period of 30 μs, and the time-domain response progress of devices with different FE) of graphene was 0.25 eV. Based on this Fermi-level, we studied the performance matrix of MRR type TO switches with different design parameters, as depicted in clh with cd being 2.6 μm and 7 μm, respectively, and the propagation loss from the metal contact could be neglected if the distance between two electrodes is larger than 2.5 μm. Thicker cladding is preferred for less propagation loss, but it will sacrifice the heating effect. Thus, if we want to design an optimal heater structure with graphene, a compromise should be made between the heater loss and heating efficiency. Here, we can notice that TO switches could achieve a higher heating efficiency and faster response if the distance between the graphene and the waveguide and the distance between the two electrodes are close enough. However, the overall performance of TO switches will also be limited by the absorption from graphene heaters and the metal electrodes. Although metal absorption is much larger than graphene, graphene is still a lossy material if its Fermi level is low. Q factor and the FWHM of the resonance peaks and consequently the power consumption. The power consumption for π = FWHM×Pπ/η and the relation between the total Q factor and the FWHM is expressed byrλ is the resonance wavelength. To obtain the best performance, the ring resonator should work in the critical coupling condition where the intrinsic Q factor equals the external Q factor:Q factor can be obtained byα is the loss of the ring, including the absorption of the graphene gα and the waveguide loss wgα (1.62 dB/cm). The gn is the group index of the fundamental TE mode. The loss of the resonator is expressed by:For the ring resonator, the loss directly impacts the FWHM of resonator covered by graphene with the same Fermi level (0.25 eV) and length as our device’s with different clh and cd can be obtained and depicted in gn is 3.68 and rλ is 2022.74 nm. Thicker cladding leads to a narrower FWHM, implying that a smaller wavelength shift is necessary to achieve effective modulation. Based on the FWHM, combined with the efficiency η, power consumption πP was calculated and shown in FOM, which is equal to 1/(π·τP), could be obtained, and they are plotted in Therefore, based on Equations (5)–(8) , and theFWHM of the resonance peak and the FOM of MRR type TO switches. Fortunately, the loss of the graphene decreases significantly with the doping level of graphene (FE), as shown in FE = 0.43 eV  could be achieved with a 371-nm cladding thickness and 2.25-μm contact distance.As discussed before, the loss induced by graphene could significantly influence the hievable ). Thus, FOM in −1μs−1 is already an ultrahigh value. Hence, graphene TO devices would allow much larger fabrication-error tolerance to achieve high performance than other technologies. Relatively high FOM can be obtained with thin cladding, and the current device design can achieve an extremely high FOM of 2.177 mW−1μs−1.As mentioned, the heavily p-doped graphene heater owns obviously smaller absorption loss, and therefore the minimum FOM of a switch with a metal heater could also be evaluated. The red curve in FOM of a 50-nm-thick gold heater MRR TO switch with the same cladding thickness and contact distance is only 0.005 mW−1μs−1, which is three orders of magnitude less than that with graphene heaters. As known, the absorption coefficient for the metal increases with the wavelength. Thus, metal heater should be placed much further away from the waveguide core in the mid-infrared range. On the other hand, graphene could become transparent in the mid-infrared wavelength range through tuning its Fermi level. Thus, the FOM of graphene-based TO switches could be much higher than those based on metal heaters. Graphene will become an exceptional nanomaterial for future MIR photonic circuits.Using these models, the FOM can be as high as 2.644 mW−1μs−1, which outclasses reported works. The doping engineering can make graphene an almost transparent heater in MIR waveband, which can be directly placed on the waveguide. Such unparalleled performance can hardly be achieved by resorting to metal heaters or doped silicon heaters. Our work proves that graphene heater owns exceptional advantages compared to other kinds of heaters, and graphene is a superb photonic material for future photonic integrated circuits working at MIR waveband.We demonstrated MRR-based and MZI-based TO switches operating at 2 μm waveband for the first time, where single-layer graphene was adopted as the heater. For the MRR device, the modulation efficiency was 0.11 nm/mW, corresponding to minimum power consumption of about 5 mW. The 10–90% rise time/90–10% fall time is 3.7/3.96 μs. Such dynamic response is close to the fastest silicon TO devices ever reported. As for the MZI-based device, an insertion loss of 2.0 dB and an outstanding extinction ratio of 27.08 dB with broadband operation wavelength range was achieved. The switching time was also characterized to be 4.92/4.97 μs respectively. The insertion loss could be minimized by the chemical doping of graphene, and the relatively high power consumption can be largely suppressed by a more appropriate design of contact distance. The numerical analysis showed that the performance of the graphene-assisted TO switch can be improved significantly by appropriately designing the structure of the heater and doping the graphene. The


By transferring graphene layers to plasmonic nanoparticles immobilized on a glass substrate, the scattering profiles of the nanoprobes were adjusted in terms of the position, width, and intensity of the peaks to determine the optimal conditions for measuring the PRET signal. Using the optimized graphene-covered plasmonic nanoprobe, we obtained calibration curves over a wide concentration range from femtomoles to millimoles for hydrogen peroxide based on the change in the PRET signal. Before monitoring cellular ROS, we confirmed that a high density of cells adhered well to the graphene-covered plasmonic interface by observing immunofluorescence images of the cytoskeleton of the immobilized cells. Finally, we monitored the real-time ROS generated by the cells under oxidative stress conditions by directly measuring the spectral changes of the probes around the cells. We believe that the proposed graphene-covered tunable plasmonic interface has versatile applicability for investigating cellular stress and disease progression by monitoring ROS levels under various cellular conditions.Reactive oxygen species (ROS) regulate various physiological and pathological conditions in cells by interacting with signaling molecules and inducing oxidative stress. Therefore, sensitive monitoring of ROS levels in living cells is important to track cellular state and study the complex role of ROS in the development of various pathologies. Herein, we present an optically tunable plasmonic interface covered with graphene to monitor cellular ROS levels with superior sensitivity and cellular comfortability. As a sensing principle, we employed plasmon resonance energy transfer (PRET)-based spectral quenching dips modulated by redox-active cytochrome The online version contains supplementary material available at 10.1186/s40580-022-00315-9. Reactive oxygen species (ROS), like superoxide anions, hydrogen peroxide, and hydroxyl radicals, are molecules with strong oxidative power and high reactivity that regulate a variety of biological processes –4. In noc (Cyt c) induces unique spectral quenching dips in the scattering profile of the plasmonic probe, which can be changed by the presence of ROS. Before cellular ROS monitoring, it would be better to use optically adjustable and biocompatible interfaces for monitoring oxidative stress in cells. Because the PRET signals stem from the spectral overlap between the scattering of the plasmonic nanoprobe and the absorption of the redox-active Cyt c, fine-tuning of the scattering spectra of the probes is essential to achieve high sensitivity. Moreover, a comfortable interface should be provided to cells to monitor the oxidative stress experienced under certain extracellular or intracellular conditions. Recent reports have shown that diverse cell types, including fibroblasts [Various methods have been developed to detect ROS generated by cells using fluorescence –27, chemroblasts , neuronsroblasts , and ostroblasts , adhere c reacted with ROS. By measuring PRET signals in real time, we can quantitatively detect ROS levels and monitor cellular ROS.In this study, we demonstrate a plasmonic interface covered with graphene layers to optically monitor the ROS generated from living cells. As illustrated in Fig. 2SO4, 95%), hydrogen peroxide , ammonium persulfate (98%), ethanol (99.9%), dimethyl sulfoxide (99.8%), acetone (99.5%), 2-propanol , and nitric acid (69%) were purchased from Samchun Pure Chemical Co. Ltd. . Silver nanoparticle , cytochrome c (Cyt c) from the equine heart (≥ 95%), l-ascorbic acid (AA), 3-aminopropyltriethoxysilane (APTES), phosphate-buffered saline (PBS), poly-l-lysine (PLL) solution (0.01%), sodium (meta) arsenite (≥ 90%), Triton X-100, bovine serum albumin (BSA), and Hoechst 33258 were purchased from Sigma-Aldrich . Gold nanoparticles  were purchased from BBI solutions . The human dermal fibroblast cell line (HDF) was obtained from NeoRegen Biotech Co., Ltd. . Dulbecco’s modified Eagle medium (DMEM), penicillin/streptomycin (Penstrep), and 0.05% trypsin-EDTA were purchased from Gibco-Life Technologies . Fetal bovine serum (FBS) was purchased from MP Biomedicals . Formaldehyde (4%) and Alexa Fluor 488 phalloidin were purchased from Thermo Fisher Scientific . Rabbit monoclonal anti-α-tubulin and Alexa 647-conjugated anti-rabbit secondary antibodies were purchased from Abcam .A poly(polydimethylsiloxane) (PDMS) elastomer kit (Sylgard 184) was purchased from Dow Corning . Copper (Cu) foil  was purchased from Alfa Aesar . Sulfuric acid .4 and H2 as precursor gases. The substrate was first annealed at 1000 ℃ for 30 min under an H2 flow (100 sccm). Subsequently, the carbon source gas, CH4 (10 sccm), was introduced into the quartz tube. After 30 min of growth, the CH4 gas was turned off, and the copper substrate was removed from the heating area of the furnace to cool down at room temperature under H2 flow.Before graphene growth, the Cu foils were cut into 10 cm × 10 cm pieces. Nitric acid was diluted in deionized (DI) water under stirring. The Cu foils were immersed in dilute nitric acid for approximately 30 s. The cleaned Cu foils were successively washed with isopropanol, acetone, and DI water three times. Monolayer graphene was then grown on the cleaned copper foil via a CVD system using CHTEM analysis was performed to characterize plasmonic NPs. To prepare the specimens, 10 µL of each NP solution was dropped onto a carbon-coated 300-mesh TEM grid . TEM images were obtained using a transmission electron microscope  operating at an acceleration voltage of 120 kV.2SO4/H2O2 = 7:3 v/v) for 1 h, rinsed with ethanol and DI water, and then dried in a stream of N2. The cleaned glass slide was immersed in an ethanol solution of 1 mM APTES for 24 h. The surface modified with APTES was washed with pure ethanol and dried in a stream of N2. To immobilize plasmonic NPs on the glass slide, 200 µL of colloidal NP solution was dropped onto the APTES-modified glass slide for 30 s and washed with DI water to remove excess NPs. Prior to transferring the graphene layers onto the NP-immobilized glass substrate, the graphene was floated in a 1 M ammonium persulfate aqueous solution for 6 h to completely dissolve the Cu foil. Then, it was washed twice with DI water for 15 min and washed with HCl for 10 min at room temperature (RT) for complete etching of Cu. After the etched graphene was placed on a glass slide, the PMMA layer was removed with acetone and IPA.The glass slide was treated with piranha solution  at an accelerating voltage of 10 kV.The surface topologies and height profiles of the transferred graphene layers were also obtained using an AFM  in the non-contact mode.A micro-Raman system combined with a spectrometer SR-303i (Andor Technology) and a 50 mW 532-nm laser module PSU-III-FDA  was used for the characterization of graphene. The system was comprised of an integral Olympus BX51 microscope with a 20× objective lens. The Raman spectra were collected at an exposure time of 1 s (five accumulations).Dark-field scattering imaging of graphene-covered plasmonic NPs according to the number of graphene layers and the corresponding spectral analysis under each condition were performed using a dark-field microscope  equipped with a hyperspectral imaging spectrophotometer . A 20× objective lens was used for imaging, and the integration time for collecting the scattering spectra was 0.3 s. For all data, the reliabilities of the measured intensity, spectral shifts, and full width at half maximum (FWHM) were evaluated based on the standard deviation from 60 different nanoprobes.The photoluminescence (PL) properties of the plasmonic NP-graphene interface were characterized using a fluorescence spectrometer  with an excitation picosecond laser at 520 nm in the range 520–900 nm.2O2 concentration were measured using the above-mentioned dark-field-based hyperspectral system. Prior to the measurement of the quenching dips, the intrinsic spectra of the single NPs were measured under DI water in the PDMS well. To measure the initial spectra of the NPs before exposure to H2O2, DI water in the reaction well was replaced with a reduced Cyt c aqueous solution as a redox probe, which was prepared by preincubation with 100 µM Cyt c and 50 mM AA (5:1 v/v) for 1 h. Then, H2O2 was injected into the PDMS well, and the ratio between the reduced Cyt c and H2O2 was set to 5:1 v/v. After a 2 min reaction, the scattering spectra of the single NPs because of H2O2 exposure were collected. All collected spectra were normalized, and changes in the spectral quenching dips were measured before and after H2O2 exposure. For all the data, the reliability of the observed spectral shift and intensity was examined using the standard deviation of 60 different NPs.To demonstrate PRET-based ROS quantification, changes in the quenching dips of the single plasmonic NPs depending on the HTo examine the biocompatibility of the plasmonic NP-graphene interface, cells incubated for 24 h were fixed with 4% formaldehyde in PBS for 15 min at RT and permeabilized with 0.4% Triton X-100 for 15 min. The cells were washed twice with PBS for 15 min. For imaging microtubules, after blocking the cells with 5% BSA in PBS for 1 h, cells were incubated with a rabbit monoclonal anti-α-tubulin antibody  overnight at 4 ℃ and washed five times with PBS for 2 min over 5 times. The cells were further incubated with an Alexa 647-conjugated anti-rabbit secondary antibody  for 1 h at RT. F-actin was stained with Alexa Fluor 488-phalloidin (Invitrogen) for 1 h at RT. Nucleic acids were stained with the DNA-binding dye Hoechst 33258 . Stained cells were visualized using a confocal laser scanning microscope .3 cells/200 µL) and A375P (5 × 103 cells/200 µL) were seeded in 0.01% PLL-treated plasmonic NP-graphene interface and incubated at 37 °C and 5% CO2 atmosphere for 24 h. Then, 0.2 M NaAsO2 and reduced Cyt c diluted in DMEM were injected onto the plasmonic NP-graphene interface in the PDMS well. The change in the scattering spectra induced by the ROS was monitored using a dark-field-based hyperspectral system.HDF cells . After incubation with the toxicants for 1 h, the cells were washed with PBS and exposed to 20 µM 2,7-dichlorofluoroscein diacetate (DCFDA) diluted in DMEM. After incubation for 50 min at 37 °C in the dark, the cells were washed with PBS and imaged using a confocal laser-scanning microscope.Cells were seeded at 0.01% PLL-treated plasmonic NP-graphene interface and incubated at 37 ℃ in a 5% COc in this study) are near the plasmonic nanoprobes. That is, the PRET signal is a result of light energy transfer from plasmonic nanoparticles (NP) to light-absorbing molecules [c. As summarized in Fig. c were clearly observed without omitting or truncating the dips. This revealed that there is an optimal condition for measuring the Cyt c-mediated PRET signal in terms of the position, width, and intensity of the scattering peaks of the plasmonic nanoprobes. When we layer the optically transparent and ultra-thin graphene—an excellent electrical conductor—onto plasmonic nanoprobes, we expected that electron transfer between them can modulate the scattering properties of the probes according to the type of plasmonic NPs, as shown in Fig. In principle, PRET signals are measured in the form of spectral quenching dips in the scattering spectrum of the plasmonic nanoprobe matching with the molecular absorption bands when the light-absorbing molecules (Cyt olecules –47. We nsc) of the nanoparticles was calculated by varying the sizes of the SNP and GNP and the number of graphene layers. For example, diameters of 100, 110, and 120 nm were considered for SNP, and diameters of 50, 55, and 60 nm were considered for GNP. In the case of graphene layers, 0–4 layers were considered . The scattering cross-section  and bi-layered graphene (~ 2 nm). Additionally, we measured the Raman scattering to further confirm the presence of the graphene layer in the plasmonic NP-graphene interface. As shown in Fig. − 1 (G peak) and 2670 cm− 1 (2D peak), which are well known as characteristic peaks of graphene [To prepare the plasmonic NP-graphene interface, plasmonic NPs were first immobilized on an amine-functionalized glass substrate, followed by transferring the graphene layer onto the immobilized NPs, as illustrated in Fig. graphene , 52. Thegraphene , 53. TheWe next investigated the optical tunability of plasmonic NPs by increasing the number of graphene layers. Dark-field scattering images showed that the scattering colors of the SNP probes gradually changed from green to red as the number of graphene layers increased Fig. e. Figurec was evaluated using the two types of plasmonic NPs (SNP and GNP) and their hybrid interfaces covered with different numbers of graphene layers. As expected, the quenching dips by PRET were the largest when the scattering peak of the nanoprobe exactly matched the absorption wavelength of Cyt c  was calculated as 2 fM, which is six orders of magnitude lower than that of the commercially available ROS assay kit (ca. 1 µM) [c, as described in Fig. For ROS detection, we intentionally prepared fully reduced Cyt ed Cyt c . ROS-ind8 M Fig. f. The SNa. 1 µM) . This isa. 1 µM)  and Au-Pa. 1 µM)  without Before monitoring the ROS in living cells, cellular adhesion on the plasmonic SNP-graphene interface was examined using immunofluorescence staining for F-actin (green), α-tubulin (red), and nuclei (blue). Human dermal fibroblasts (HDF) and human melanoma cell lines A375P) were seeded on the prepared SNP-graphene interface and on a glass slide (as a control) , oxidative stress-induced cells , and cancer cells , as displayed in Fig. Having demonstrated that the graphene-covered SNP serves as an excellent probe for ultrasensitive detection of ROS and provides a comfortable interface to cells, we next monitored the ROS generated from living cells in real time. The reduced Cyt i.e., A37P, as disi.e., A37P, as dis2 clearly shows that the change in the quenching dip at 550 nm is greater than that of the unexposed HDF. Within 10 min, ROS levels were measured to the ranges comparable to the 10 nM–10 µM H2O2 for the three cases, when estimated from the calibration curve. After 60 min, the extracellular ROS levels were more clearly distinguishable according to the cellular conditions, with ROS levels comparable to 1 mM, 100 mM, and over 100 mM H2O2 for HDF, NaAsO2-exposed HDF, and A375P, respectively. Through our real-time measurements, we observed that stressed cells released ROS more rapidly into the extracellular environment than normal cells. The observed difference in ROS release kinetics is consistent with the fluorescence intensity for intracellular ROS production measured after additional incubation with a ROS-indicating dye, 2,7-dichlorofluoroscein diacetate (DCFDA)  and number of graphene layers . We observed enhanced Cyt c-mediated PRET signals by modulation of electron movement through the interface between the NP and graphene. Using the optimized probes, we detected H2O2, a representative ROS, at the femtomolar level, which is six orders of magnitude lower than that of the commercially available ROS assay kit (ca. 1 µM). In addition, we demonstrated that a plasmonic-graphene hybrid interface provides an optically excellent and comfortable surface for real-time monitoring of cellular ROS from living cells. The proposed graphene-covered tunable plasmonic interface has versatile applications for studying cellular stress and disease progression by monitoring ROS levels under various cellular conditions.We demonstrated the in situ optical monitoring of ROS generated from living cells on the plasmonic NP-graphene interface based on the redox-active Cyt Additional file 1: Figure S1. Simulation of scattering spectra according to the number of graphene layers on SNP. (a) 110 nm SNP. (b) 120 nm SNP. (i) Full spectrum. (ii-iv) Plots showing shifts of λmax (ii), FWHM (iii), and scattering cross-section (σsc) (iv). Figure S2. Simulation of scattering spectra according to the number of graphene layers on GNP. (a) 55 nm GNP. (b) 60 nm GNP. (i) Full spectrum. (ii–iv) Plots showing shifts of λmax (ii), FWHM (iii), and scattering cross-section (σsc) (iv). Figure S3. Representative TEM images of the used SNPs. The average size (for n = 40) was observed to be 101.6 ± 5.0 nm . Scale bars represent 25 nm. Figure S4. TEM images of the used GNPs. Average size (for n = 40) was 49.5 ± 2.6 nm. Scale bars represent 25 nm. Figure S5. Scattering properties of the plasmonic GNP-graphene interface. (a) Dark-field scattering images of the graphene covered-plasmonic GNP with increasing number of graphene layers. The scale bars represent 10 µm. (b) Corresponding scattering spectra measured for the GNPs with increasing number of graphene layers. (c) Plots for the shifts in terms of λmax (i), FWHM (ii), and intensity (iii) with increasing the graphene layer on the GNP. Figure S6. Changes in photoluminescence (PL) intensities of graphene-covered NPs. (a) SNP. (b) GNP. (i) Schematic diagram, (ii) PL spectrum, and (iii) Plot for the change in PL intensity of NP at 550 nm in the presence of graphene layer. Figure S7. Fluorescence images of intracellular ROS in cells. (a) HDF, (b) NaAsO2-exposed HDF, and (c) A375P. The green fluorescence indicates intracellular ROS visualized by staining with a ROS indicating dye, 2,7-dichlorofluoroscein diacetate (DCFDA). The scale bars represent 50 µm.


Studies on depositions of chemical vapour deposition (CVD) diamond films have shown that flame combustion has the highest deposition rates without involving microwave plasma and direct current arc. Thus, here we report on our study of few-layer graphene grown by flame deposition. A horizontal CVD reactor was modified for the synthesis of flame deposition of few-layer graphene on a Cu substrate. It was found that graphene obtained has comparable quality to that obtained with other flame deposition setups reported in the literature as determined from Raman spectroscopy, sheet resistance, and transmission electron microscopy. Calculation of the chemical kinetics reveals a gas phase species that has a close correlation to the growth rate of graphene. This was further correlated with van't Hoff analysis of the reaction, which shows that the growth reaction has a single dominating mechanism for temperatures in the range of 400 °C to 1000 °C. Arrhenius analysis also was found to be in good agreement with this result. This study shows few-layer graphene growth proceeds through different pathways from a CVD grown graphene and also highlights flame deposition as a viable method for graphene growth. Growth kinetics of few-layer graphene grown by flame deposition correlates to BIN20J species in a methane to soot mechanism model. One of them is that due to its batch production coupled with long reaction times at high temperatures, CVD becomes a very energy intensive method equating to increased costs. Through this method, graphene with resistivity as low as 143 Ω cm has been achieved.2 Bae et al.3 growing graphene films reports a sheet resistance of 30 Ω per square. In comparison, pristine graphene obtained from exfoliation of graphite has 10−6 Ω cm resistivity4 with a maximum current density5 larger than 108 A cm−2.Current production of high-quality large-area graphene can only be achieved through chemical vapour deposition. No other method comes close to producing the quality of graphene produced by this method.6 Most recently, gallium and cobalt–copper alloy substrates were shown to grow graphene at lower temperatures.7,8So far, much progress has been made to reduce its energy consumption through the development of better catalysts and growth methods. In general, copper has been the choice substrate for graphene growth due to its wide availability. When compared to other elements, it is cheaper compared to other metals but its high energy barrier creates a need for high reaction temperatures for graphene to grow.9,10 However, the need for a high-power plasma generator and a good vacuum system makes the setup very complex and has high energy consumption. Thus, graphene grown by this method is very expensive due to its batch production capabilities and very strict control of the growth conditions.While such catalytic substrates could potentially reduce the reaction temperature and improve reaction rate, there is still a need for better production methods as it will ultimately determine the scale of which graphene can be produced. For now, modifications to the conventional CVD methods have been shown to allow good quality graphene to be produced at even lower temperatures. Usage of plasma has been widely reported to have reduced the growth temperatures of graphene and commercially manufactured reactors are already available for plasma enhanced CVD (PECVD). Instead of thermally activating the reactants within the gas phase or on the substrate, reactants are first turned to active species within the plasma itself. As a result, growth temperature could be lowered as low as <420 °C while maintaining a high-quality growth.11 Previous to graphene synthesis by flame deposition, Wal et al. reports of the growth of carbon nanotubes by flame deposition only requiring reaction times in the order of milliseconds.12 The combustion itself raises the temperature allowing the remaining hydrocarbons to form the desired product. One of the earliest reports of graphene grown by flame deposition used a dual flame setup.13,14 Nickel substrate was kept within a flame sheath to prevent oxidation while another carburizing flame heats and provides carbon to the substrate. The authors reported that graphene synthesized through this method did not have full coverage and contains many defects. Another group built a micro combustor for the flame deposition of graphene on Cu and Ni wires where they found high Reynolds number yields better graphene qualities.15 A setup developed by Memon et al.16 for large area graphene growth used an inverse diffusion flame. The inverse diffusion flame getters excess O2 and provides a stable flame. They reported a graphene deposit which has a monolayer ratio (I2D/IG) of 0.77 to 0.59 and defect ratio (ID/IG) of 0.35 grown at 1000 °C for 10 minutes. Our group have also reported on achieving similar quality graphene using a horizontal CVD reactor modified for flame deposition.17 This highlights the potential that this method possesses for producing graphene at lowered temperatures.Here, we explore an alternative way which could emulate plasma enhanced CVD using flame deposition. Historically, deposition of diamond by flame combustion have been widely studied and it has been shown to have a very high deposition rate when compared to other methods except for direct current arc.18 there are no known studies relating to growth mechanisms for graphene grown by flame deposition. However, combustion reactions of hydrocarbon have been studied in relation to diamond film formation.19,20 In fact, there are various models that have been built which even allows predictions of the resulting equilibrium species. In this study, we aim to study the growth kinetics of graphene grown on Cu substrate using a flame deposition method. Complimenting the experimental results are simulations of equilibrium species based upon a soot production model.21While there are many discussions on the growth mechanism for CVD grown graphene,2) gas enters from the ceramic nozzle to mix with methane (CH4) and hydrogen (H2) gas entering from a separate ingress. 2 R-type thermocouples were used to regulate and observe the temperature. One was placed with the substrate and another outside of the quartz tube within the isothermal region.Graphene was grown using a homemade horizontal hot-wall furnace fitted with a ceramic nozzle to allow combustion. The furnace core is a ceramic tube wrapped with Kanthal wires and insulated with ceramic fibres . An ignition system was attached to the nozzle to initiate combustion as shown in Substrate preparation before the deposition is as follows. A copper foil  was polished and cleaned thoroughly before use. First, various grits of sandpaper were used until reaching #2000 grit. Next, alumina polish  was used as the final finishing before cleaning with acetone  followed by distilled water in an ultrasonic bath for 30 minutes. Once done, it was dried thoroughly and stored in a dry place. For the deposition, the foils were cut into 1 cm by 1.5 cm.2 atmosphere with a flow rate of 100 sccm. It was then removed from the furnace hot-zone and allowed to cool under Ar gas flow till room temperature. Once cooled, the substrate with Ar gas, CH4 and H2 were introduced followed by O2 gas. Upon introducing O2 gas, flame ignition was initiated, and the substrate was inserted back into the heated furnace at a temperature of 1000 °C for graphene deposition. After 10 minutes deposition, the substrate was taken out from the hot-zone and allowed to cool to room temperature in an Ar atmosphere. The whole reaction takes place at atmospheric pressure and its overall procedure is shown in Before deposition, an annealing step was performed at 1000 °C for 20 minutes in a H4, O2, and H2. Partial pressure for each reactant was changed while keeping the remaining 2 reactants at the same ratio to each other. Graphene deposits obtained were transferred and characterized.3 parameters were tested, which are partial pressures of CHRs was then converted to deposit thickness, t by the following ρ = 4 × 10−5 Ω cm) was used.Graphene deposits on Cu substrate were transferred using methyl methacrylate found commonly in super glue with tissue fibres as a reinforcing matrix. Sheet resistance measurements were done on these transferred deposits using a 4-point co-linear probe with a potentiostat . Sheet resistance, 22Additionally, graphene was also transferred to a silicon wafer and copper mesh grid  for analysis by Raman spectroscopy  and transmission electron microscopy (TEM) . Fast Fourier Transform analysis was also done on bright field images from TEM using FIJI software.23 set to conditions similar to those stated in the experiments using a mechanism file containing 296 species.21Apart from experimental data, numerical analysis of the concentrations of equilibrium species was also done using Cantera4 partial pressure, P(CH4) while H2/O2 ratio was set to unity results in thicker deposit as P(CH4) increases. However, deposit thickness reaches a maximum between 0.6 and 0.8 atm P(CH4). For the dependency of P(O2), while CH4/H2 is set to unity, deposit thickness reaches a maximum at P(O2) = 0.2 atm before decreasing again as seen in P(H2). However, it should be noted that the increase in deposit thickness due to P(H2) is very small when compared to changes due to P(CH4) and P(O2). For P(H2) deposit thickness increased from 0.5 Å to 2.1 Å whereas, graphene thickness increased by 100 fold for P(CH4) and 10 fold for P(O2). In general, conditions that yields thin deposits have a larger deviation which gets smaller as the deposits get thicker. Unsurprisingly, thinner deposits are harder to reproduce as any slight changes induces a significant change in thickness. This is clearly demonstrated in Deposit thickness calculated from the measured sheet resistance is shown in P(CH4), the low initial P(CH4) coupled with high P(O2) leaves only a small amount of unreacted hydrocarbon to serve as the precursor for graphene. As it increases, there are more excess hydrocarbon allowing for thicker deposits. In P(O2) was varied, even a small addition of O2 was found to promote thicker deposits until a certain threshold which is in line with reported observations.24 It was noted that O2 was found to assist in the pyrolysis of CH4 which improves growth rate but higher P(O2) instead oxidizes and depletes aromatic hydrocarbons vital for the production of solid carbons.For 4 partial pressure was done and TEM analysis was performed on the deposit grown at CH4 : O2 : H2 = 0.8 : 0.1 : 0.1 atm which is shown in ID/IG) and monolayer ratio (I2D/IG) in P(CH4) yield deposits that are higher in defects and thicker which is in good agreement with thickness calculated from sheet resistance P(CH4). Again, a transition point was observed at the 0.4 atm to 0.5 atm range similar to the one in sheet resistance where thickness growth rate undergoes a change. Growth rate slows down beyond this point as O2 and H2 get further diluted by CH4 which hints of an optimal O2 and H2 composition in aiding the growth of graphene.Additionally, Raman characterization of deposits for the dependency on CH25 Observing another region with spherical carbon nodules yields an FFT image with rings and devoid of any spots. Raman spectra of the same deposit in −1 was detected even on blank Cu  values also shows few-layer graphene as observed by Raman analysis in TEM analysis of the deposit reveals the presence of graphene structures and amorphous carbon. Fast Fourier Transformation (FFT) of a region with moiré pattern in u Fig. S1 without 27 The absence of d = 0.213 nm diffraction spots by graphene indicates that the deposit is few-layer graphene.25 Transferring the graphene on a Cu mesh introduces additional crumples and folds which will lead to regions of multilayered polycrystalline graphene which could be concluded from the twisted diffraction rings. Such defects are not originally present on the deposit itself. Overall, we could conclude that the deposit obtained contains amorphous carbon, multilayer polycrystalline graphene as well as diamond-like carbon.Analysis of diffraction patterns of the bright spots in 4, O2, and H2 were performed using Cantera. Focussing on the effects of changing P(CH4), 2O, CO2, CO, and CH3O reach a peak at around P(CH4) = 0.5 atm and declines with higher P(CH4) since there is less O2 available. Formation of complex hydrocarbons (C6H6 and C8H10), H2 and H* radical undergoes a sharp increase as it transitions from a fuel lean mixture to a fuel rich mixture at P(CH4) = 0.2 atm. At higher concentrations, hydrocarbon species also increases but slow down after about P(CH4) = 0.5 atm. The growth rate of graphene observed in Calculations for the concentration of equilibrium species for each dependency on CHP(O2) and its equilibrium species concentration do not present any notable similarities. However, as expected, P(O2) > 0.5 atm as the gas composition turns into a fuel-lean mixture. Also, the point P(O2) = 0.2 atm where deposit thickness was highest was also the point where most hydrocarbons undergo a sharp decline in equilibrium concentration as O2 increases. Judging from the increased concentration of combustion products, oxidation reactions are the cause for this. Whereas according to P(H2) even as initial CH4 gets reduced due to Le Chatelier's principle to counter the increased P(H2). Deposit thickness in P(H2). Regardless, equilibrium species considered here does not closely follow the changes in deposition thickness from experiments. It is probable that there are other species that have a better correlation to our experimental data.Comparisons between dependency of graphene thickness on 12972032H1622016 (codename: BIN20J) is a possible candidate as it possesses the best correlation to our experimentally obtained data as shown in In order to find the chemical species vital for graphene growth, each species available in the model was evaluated. From all the species contained within the mechanism, it was found that a chemical species CP(CH4) and P(O2) in st order reaction which explains the close correlation of BIN20J concentration to deposit thickness. Calculations on the yield and selectivity of BIN20J was also done  increases as more hydrocarbon by-products are also produced at higher methane concentration. Selectivity for BIN20J increases above P(O2) = 0.2 atm but its yield reaches a plateau at that point. This is believed to be due to its consumption by excess oxygen despite its increased selectivity. Changes to deposit thickness in relation to P(H2) shows poor correlation to the predicted values of BIN20J. However as noted previously, changes in deposit thickness due to H2 dependency were an order of magnitude smaller than those due to CH4 and O2. Studies have noted that graphene could still be obtained by flame deposition even without H2 gas being used28 and chemisorbed hydrogen on Cu functions as a catalyst for CH4 heterogenous dissociation on Cu.29 From this, we believe that H2 has a small influence in gas phase kinetics and instead has a bigger role in affecting the substrate itself directly. Since the model involves only gas phase reactions, such heterogeneous reactions are left unaccounted for thus leading to the inconsistency between experimental and predicted model. Regardless, the predicted values of BIN20J has been shown to have a good correlation to experimental data.The equilibrium concentration for BIN20J displays a direct correlation to deposit thickness for e Fig. S3, it show30 An Arrhenius plot  that is less affected to the gas phase kinetics.In −1) which is markedly lower than graphene grown by conventional CVD methods as seen in 4 as determined by ab initio studies33 and mass transport is responsible for the rate-limiting step at APCVD34,35 which generally leads to an increased energy barrier  closest to our experimental values.45,46While a thorough understanding of the growth mechanism of graphene by flame deposition still eludes us, our results provides certain insights on the growth of graphene by flame deposition. First, graphene growth follows closely with the BIN20J formation using a soot formation mechanism reported by Ergut I2D/IG) of 0.65 and defect ratio (ID/IG) of 0.58 which was comparable to literature. We also show that numerical analysis by an open source software using an appropriate mechanism model provides a close prediction to the actual graphene deposition. Changes to deposit thickness when CH4 and O2 were varied was found to agree with simulated values for BIN20J. Deviations between experimental and simulation results for P(H2) suggests H2 has a minor role in gas phase kinetics and influences the heterogeneous reaction on the substrate which is not accounted for by the mechanism file used. At this current stage only the gas phase mechanism was considered but with proper improvements, a model that could predict the actual deposition is certainly possible. Considerations of the growth kinetics lead us to believe that the growth mechanism includes the gas phase formation of BIN20J followed by its adsorption on Cu substrate which then grows into graphene.An experimental flame deposition setup has been built for the growth of few-layer graphene. Raman analysis shows multilayer graphene with a monolayer ratio (There are no conflicts to declare.RA-009-C9RA01257E-s001