A surface plasmon resonance (SPR) sensor based on gate-controlled periodic graphene ribbons array is reported. Different from the conventional methods by monitoring reflectivity variations with respect to incident angle or wavelength, this approach measures the change in SPR curve against the variation of graphene chemical potential  at both fixed incident angle and wavelength without the need of rotating mirror, tunable filter or spectrometer for angular or wavelength interrogation. Theoretical calculations show that the sensitivities are 36,401.1 mV/RIU, 40,676.5 mV/RIU, 40,918.2 mV/RIU, and 41,160 mV/RIU for analyte refractive index (RI) equal to 1.33, 1.34, 1.35 and 1.36; their figure of merit (1/RIU) are 21.84, 24, 23.74 and 23.69, respectively. Significantly, the enhancement in the non-uniform local field due to the subwavelength graphene ribbon resonator can facilitate the detection in redistribution of protein monolayers modeled as dielectric bricks. Optical biosensors monitor variations of the scattering characteristics of incident light including intensity, phase, wavelength, and angle due to interaction between the target analyte and bioreceptor such as nucleic acid sequences, proteins, antibody, enzymes, and etc. Optical fiber with a tapered small core for single-mode operation designed to serve as a high sensitivity RI sensor was reported2. The resonant waveguide gratings were extensively reviewed3, particularly including applications of sensors for detecting the RI change on the waveguide grating surface.The emergence of the respiratory virus COVID-19 outbreak allows us to know the unprecedentedly urgent need of a rapid and accuracy diagnostic test method. Among the known diagnostic techniques, label-free optical biosensors provide high sensitivity and robustness solutions. Most importantly, such a biosensor can be integrated into a compact device and enables the possibility of bedside (or point-of-care) testing9. Additionally, enhanced absorption of surface plasmons on a periodic array of graphene ribbons for TM-polarization  was reported13. Specifically, the obvious optical absorption due to the SPR on graphene ribbons, particularly on its fundamental resonant mode, was demonstrated10.Surface plasmons are collective oscillation of the free electron gas density on the surface of noble metals or graphene. Through the electromagnetic wave coupling between photon and plasmon, the excitation of SPPs (surface plasmon polaritons) is present with its field exponentially decaying (evanescent wave) along the direction perpendicular to the surface14. A nanodevices for detection and real-time monitoring of biomolecular events using SPR and SPR imaging were thoroughly reviewed; the signal modulation schemes including angle, wavelength, amplitude and phase were discussed, as well as their coupling mechanism15. The SPR imaging techniques incorporating wavelength scanning based on solid-state wavelength filter were reviewed from the perspective of detection speed, sensitivity and portability16. RI sensor using long-range surface plasmon mode, which exhibits symmetric field pattern, was shown to have high sensitivity compared with conventional SPR ones due to low loss17. The plasmonic nanograting was employed to tailor the dispersion of plasmonic response; more specifically, the enhancement in quality (Q) factor further improves the figure of merit (FOM) of the biosensor18. The nanoplasmonic biosensor plays an important role in optical sensors because of its high sensitivity to the change in RI. More specifically, the biomolecular recognition elements on the surface of biosensors recognize and capture analyte in a liquid sample, causing an increase in the average RI on the sensor surface; several approaches for detecting the RI change were reported23. An effective design approach for achieving the high FOM of a guided-mode resonance sensor made of a grating-waveguide was numerically studied24. Furthermore, by adding a dielectric thin film having a high real part of permittivity for dramatically improving the sensor sensitivity was reported25. In their study, the guided-wave SPR structure can enhance the evanescent field at the interface between silicon and cover. Additionally, by spin-coating the GeSe nanosheets on the surface of noble metal (Au), a SPR sensor can significantly enhance its sensitively26.Moreover, the sensed medium attached to the position where SPR occurs strongly affects the resonance effect. Such a phenomenon has been widely applied in the design of biosensors for detecting the change in RI of an analyte. To mention a few, the literature focused on SPR sensors, which were designed for detecting chemical and biological species, based on the unlocalized surface plasmons propagating along wave-guiding structures were intensively reviewed27. A transmission type SPR-based sensor made of graphene nanoribbons array in infrared (IR) wavelength range was proposed to detect the wavelength shift of the resonant transmission dip due to the RI change of the analyte medium placed on the sensor surface28. A graphene-based tunable mid-IR biosensor was demonstrated on the protein characterization including both complex RI extraction and vibration fingerprints. The gate-controlled self-biasing graphene ribbons array is dynamically tuned to selectively excite the local SPR mode at different frequencies29.Regarding the biosensors made of graphene ribbons, the researchers detected multiple SPR absorption bands on graphene ribbons by changing the graphene chemical potentialThis research reports the computational design of a biosensor for RI sensing at mid-IR wavelengths. Specifically, the SPR modes supported by graphene nanoribbons are excited by evanescent wave rather than plane wave. The excitation of SPR modes in the nanoresonators (nanoribbons) enables the local-field enhancement around the slit (the region between two adjacent ribbons) edges. The placement of analyte on the surface of nanoresonators strongly affects the resonant condition of surface plasmons, particularly on the fundamental mode. As is well known, the wavelength- and angle-interrogation are the two major approaches for detecting the RI change due to a shift in SPR-curve. Significantly, in this research, graphene chemical potential scan is employed to replace the sophisticated wavelength modulation or incident angle scan. By electronically tuning the gate voltage (dc) to change the chemical potential (Fermi energy) in graphene, one can detect the smallest resolvable RI change of an analyte through the shift in SPR curve (against the variation of gate voltage). Furthermore, the tunable gate voltage can be provided by a high-resolution digital-to-analog converter (DAC) equipped with an amplifier. Furthermore, a periodic dielectric bricks array was employed to model discrete sensing mediums such as protein monolayers. The perturbation on SPR curve due to their redistribution and variation of surface percentage coverage (concentration) were also examined. Such a mechanism can be a potential candidate for designing a cost effective biosensor with acceptable performance parameters.x-axis; they are deposited on a silica , carrier relaxation time  image detection. The detail process of graphene transfer in fabricating the crystalline silicon-graphene-gate oxide structure can be found in Fig. 9 of33. Additionally, the graphene layer between Si and 34. As was reported by the authors, this device is compatible with the conventional integrated circuits process. Regarding the issue of interaction between graphene and 35, the electronic measurement showed that the single layer graphene on 29.The single layer graphene inserted between the crystalline silicon and gate oxide has been successfully implemented in the structure of a graphene photodiode-oxide-semiconductor field effect transistor (PDOSFET) shown in Fig. 8 ofy-axis) is normally incident upon the wedge surface shown in Fig. y-axis is much greater than the operating wavelength; the electric- and magnetic-fields have no variation along that direction. Therefore, a simplified 2D electromagnetic model depicted in Fig. 36 exponentially decaying along the z-axis excites the SPR modes on the graphene ribbons. Notably, because that the period of graphene ribbons array ; therefore, it is more feasible and efficient than the commonly used methods.Figure  entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaWe redraw the reflectance against chemical potential shown in Fig. entclass1pt{minimaentclass1pt{minimaV is the voltage induced on the graphene sheet.To understand the physical insight of wave process involved in the structure, the absorption against wavelength for the same biosensor employed in the previous example is calculated subject to an arbitrary analyte RI such as x-component current density  structure has a significant sensitivity enhancement. Nevertheless, due to the non-uniform evanescent field, the developed sensor can detect the redistribution of discrete analyte as shown in the later section.Furthermore, the three wavelength-selective (resonant) absorption (in blue color) occurring at 40 by fitting a sum of exponential functions to the sampled data of Returning to Fig. To evaluate the performance of this sensor, we calculate the RI sensitivity and figure of merit. The analyte under test is modeled as a uniform dielectric layer with refractive index mentclass2pt{minimThe typical RI detection resolution is around N is the bit number of a DAC and V for Returning to Table  29  to mimic their redistribution. Figure In Fig. 41. Parameter C, in fact, can also be regarded as the protein concentration. Here, C) from 0.1 to 0.05% with a step In the second example, in each unit cell the protein monolayer is positioned at the center of slit, shown in the inset of Fig. y-axis in practical manufacture; that is, the electric- and magnetic-fields are supposed to have no variation along that direction. Due to the periodicity along the x-axis (periodic graphene ribbons array), the electromagnetic fields in each uniform dielectric layer and semi-infinite medium are represented by Rayleigh expansions (or Floquet–Fourier series)45. Owing to the zero thickness approximation of the graphene grating, discontinuity in the tangential component of magnetic fields at the interface (surface of periodic graphene ribbons array) between two uniform mediums equals to the conduction current induced on the graphene strips array. The relationship between tangential electric and magnetic fields can be established via the graphene optical conductivity. Through the conventional mode-matching method, the scattering characteristics of the graphene ribbons array can be determined; however, the Gibbs phenomenon in Floquet–Fourier series due to the discontinuity of tangential electric field causes poor numerical convergence in scattering analysis. Specifically, as was reported47, traditional approach of rigorous coupled-wave analysis experienced a poor convergence in particular under the resonance of surface plasmon-polariton wave. Therefore, local basis functions inherently satisfying the field nature in graphene ribbon and slit regions were employed to modify the input–output relation of the periodic graphene ribbons array while the whole formulation can still fit into the standard procedure of Fourier modal method12. Some of the mathematical formulations together with the numerical convergence check -selective property of the SPR curve is essential. A clear dip allows us to easily identify the RI change. In Fig. 50; however, it is apparent to see that the resonant wavelength is varying in accordance with the change in the slit width. Additionally, the line width of SPR curve is getting wide when the slit width is decreasing. This may be explained as the occurrence of strong mutual coupling between two adjacent ribbons for the case of narrow slit width. Moreover, the coupling is caused by the leakage in resonator10, reducing the quality factor (increase in line width) of SPR curve. Contrarily, the SPR curves gradually converges for In Fig. In Fig. The computational design of an evanescent wave biosensor incorporating the SPR on periodic graphene ribbons array is carried out through the electromagnetic modeling and full-wave simulation. Different from the conventional approach by measuring the shift in SPR with respect to the operating wavelength or incident angle of a TM-polarized light, the self-biasing scheme by tuning the chemical potential of graphene (via modifying applied gate voltage) is implemented to track the SPR response. Moreover, due to ultrastrong Supplementary material 1


Retinol isotope dilution (RID) is a well-accepted technique for assessing vitamin A status . Here, in an effort to increase understanding of the method, we briefly review RID equations and discuss their included variables and their coefficients . Then, we focus on contributions of another technique, model-based compartmental analysis and especially the “super-person” approach, that advance the RID method. Specifically, we explain how adding this modeling component, which involves taking 1 additional blood sample from each subject, provides population-specific estimates for the RID coefficients that can be used in the equation instead of values derived from the literature; using model-derived RID coefficients results in improved confidence in predictions of TBS for both a group and its individuals. We note that work is still needed to identify the optimal time for applying RID in different groups and to quantify vitamin A absorption efficiency. Finally, we mention other contributions of modeling, including the use of theoretical data to verify the accuracy of RID predictions and the additional knowledge that model-based compartmental analysis provides about whole-body vitamin A kinetics. Since Furr et al.  publisheIn this article, we delve deeper into RID equations and their included assumptions, with the aim of enhancing understanding of the complexities of the method. Then, we discuss how addition of a super-person modeling component leads to improved confidence in RID predictions by providing population-specific values for the coefficients. We also highlight other ways that model-based compartmental analysis contributes to RID and to our understanding of vitamin A metabolism.Isotope dilution was originally explored by Bausch and Rietz  to estimF is the fraction of a dose of orally administered deuterated vitamin A that is absorbed and retained, dose is the administered tracer dose, S is the ratio of retinol SA in plasma to that in liver after mixing of tracer and tracee, a is a correction for fraction of the dose absorbed and retained but subsequently lost via catabolism, H:D is the postmixing hydrogen-to-deuterium ratio  in plasma retinol, and −1 was described as a correction for the contribution of the dose to vitamin A stores. Furr et al.  to predict liver vitamin A in generally healthy adult surgical patients . Predict\documentF = 0.50 , a = 0.913C10]retinol kinetic data collected in healthy young adults followed for 14 d after ingestion of [13C10]retinyl acetate. The modified equation  to that in stores (SAsit) for individual i at time t. Then:It is informative to rewrite and expand si is the mass of vitamin A in stores for individual iand Msi is assumed to be constant over the duration of the RID study.where Md) either from the literature  or from a super-person compartmental model . TBS predicted for a given subject using derived coefficients are as follows:In practice, mean values used for the coefficients to estimate TBS in an individual are derived .Note that not only SAp) to account for the facts that vitamin A absorption is not 100%, that all of the absorbed dose does not exchange with vitamin A in stores before utilization, and that, because of further vitamin A utilization, not all of the dose that reaches stores is still there at the time of blood sampling; also, to predict the mass of vitamin A in stores by RID, we need S (SApdt/SAsdt) derived either from the literature or from modeling.As implied above, application of an RID equation involves adjusting the measured variable  rather than directly to tissues for utilization before the tracer has mixed with vitamin A in stores. Thus, a better descriptor for F in In addition to absorption efficiency, RID coefficients also need to account for tracer loss between the time of dosing and blood sampling. Along with fecal measurements, cumulative urinary excretion of label was determined over 4–6 d in Sivakumar and Reddy  and Aklaa as used in the Olson and isotope mass balance equations . Because tracer catabolism occurs continually, first following rapid uptake of label from plasma into tissues and later when tracer has mixed with endogenous vitamin A, it makes sense to use a composite term Fa, as in The coefficient quations  assumes S, the ratio of retinol SA in plasma to that in stores, Haskell et al. t is the fraction of the labeled dose in compartment I, Sdt is the ratio of retinol SA in plasma to that in stores, [FaS]dt is the composite RID coefficient, and M(I) is the mass of vitamin A in compartment I; note that values for F(I) and M(I) are provided as part of the model solution. Changes in the coefficients Fa and S as a function of time are shown in Fa, the value peaks at 90% of the absorbed dose at 5 d and then slowly and gradually decreases as catabolism of tracer continues. For S, the value falls to and stays below 1 once SAp crosses over SA in stores (SAs); 1 mo or so after dose ingestion, S will reach and remain at an equilibrium value (Seq) if dietary input of vitamin A is relatively constant. Through theoretical work, we have determined that the value for Seq is R/[R + R], where R   is the rilibrium .FaS]dt. This value can be used to calculate TBS for individuals using each subject's measured SAp at time t ≥4 d. We have shown using data for theoretical subjects that this method provides excellent prediction of TBS for the group  and reasonably accurate values for individuals  . As studies are done in various settings, researchers can determine the CV for FaS at various times and thus recommend when to apply RID, perhaps identifying a universal value for the composite coefficient at the time when the CV% for the composite coefficient FaS is lowest.Although it would be ideal if one could recommend an optimal time for applying RID in children and adults over a wide range of stores, that is not (yet) possible. Present knowledge suggests that, when TBS is low, RID should be done early , but when stores are high, later  is better. From modeling , we knowOverall, the capacity of modeling to provide more accurate values for the RID coefficients helps to justify the additional resources required . If investigators are planning to do a traditional RID experiment in children, it is worth considering the addition of a super-child design, which requires just 1 additional blood sample from each subject, an estimate of vitamin A intake, and a duration of 28–42 d. This will yield not only population-specific values for the RID coefficients, thus improving confidence in the group mean RID estimate of TBS, but it will also allow for reasonably good predictions of TBS in individuals . ImportaFinally, while an RID equation can readily yield a value for TBS, it is impossible to know whether that prediction is accurate—the need for a “gold standard” was highlighted by Quadro . In seve


R2 = 0.744; P < 0.001), with MS demonstrating no loss of PD-L1 protein, even in sections with significant signal loss by IHC impacting diagnostic category. Clones 22C3 and 28-8 were most susceptible to signal loss, with E1L3N demonstrating the most robust signal . Increased humidity and temperature resulted in significant acceleration of immunoreactivity loss, which was mitigated by storage with desiccant. MS demonstrated only modest oxidation of 274 methionine-containing peptides and aligned with IHC results suggesting peptide oxidation is not a major factor. These data imply immunoreactivity loss driven by humidity and temperature results in structural distortion of epitopes rendering them unsuitable for antibody binding following epitope retrieval. Limitations of IHC biomarker analysis from stored tissue sections may be mitigated by cost-effective use of desiccant when appropriate. In some scenarios, complementary MS is a preferred approach for retrospective analyses of archival FFPE tissue collections.Immunohistochemistry (IHC) using formalin-fixed, paraffin embedded (FFPE) tissue is limited by epitope masking, posttranslational modification and immunoreactivity loss that occurs in stored tissue by poorly characterized mechanisms. Conformational epitopes recognized by many programmed-death-ligand-1 (PD-L1) IHC assays are particularly susceptible to degradation and provide an ideal model for understanding signal loss in stored FFPE tissue. Here we assessed 1206 tissue sections to evaluate environmental factors impacting immunoreactivity loss. PD-L1 IHC using four antibodies , raised against intracellular and extracellular epitopes, was assessed in stored FFPE tissue alongside quantitative mass spectrometry (MS). Global proteome analyses were used to assess proteome-wide oxidation across an inventory of 3041 protein groups . PD-L1 quantitation correlated well with IHC expression on unaged sections . Increased humidity and temperature resulted in considerable immunoreactivity loss, particularly among antibodies recognizing extracellular and discontinuous epitopes, which could be partially mitigated with the use of desiccant. However, even in significantly affected tissues MS was able to quantitate PD-L1 expression and assess peptide oxidation. These results suggest MS is suited for biomarker assessments using stored sections—a sample type commonly encountered in oncology research. Formalin-fixation followed by paraffin embedding is the most widely used method for preparing and preserving tissue specimens for clinical and research purposes world-wide. Obtaining, handling, and storing formalin-fixed, paraffin embedded (FFPE) tissue sections presents unique challenges to global pharmaceutical research and development. Standard tissue collections, often influenced by geography and local regulations, can be limiting to the extent that only sectioned tissues adhered to glass slides are available for analysis. This impacts robust biomarker development, since stored FFPE tissue is susceptible to degradation that alters both protein and nucleic-acid assays –5. RecenPrevious studies have found antigen degradation to be both antigen specific and condition dependent; therefore, a detailed understanding of how specific handling and storage conditions affect novel assays warrants greater appreciation in earlier stages of development. FFPE tissue sections are more prone to loss than tissue blocks , 15 and An alternative approach to IHC for protein quantitation in archival tissues is analysis by targeted mass spectrometry (MS) –27. ThisHere we describe a reproducible model of accelerated antigen instability testing and use it to address questions of how immunoreactivity is affected in stored tissue. We specifically evaluate the role of protein oxidation in antigen degradation, how different model IHC assays respond to accelerated conditions, and how MS may be used to evaluate and overcome the limitations of IHC in detecting PD-L1 in stored FFPE tissue sections in the research setting.FFPE tissue blocks of non-small cell lung carcinoma (NSCLC), gastric carcinoma, placenta, and tonsil tissue were commercially acquired from Asterand Bioscience , US Biomax , Tristar Technology Group , and Indiana University Health Methodist Hospital biobank  in either tissue microarray (TMA) or whole section format. Tissue blocks were obtained between 2012 and 2018. Some 1206 tissue sections were evaluated from 35 gastric carcinomas [tubular adenocarcinoma], 10 NSCLCs [2 primary squamous cell carcinoma (SCC) and 8 primary adenocarcinoma (ADC)], 6 tonsil, and 6 placenta samples. Unless otherwise stated, microtomy was performed immediately prior to experiment initiation. Normal storage conditions refer to a monitored and controlled laboratory environment with a relative humidity range of 14.4–80.5% (average 46.8%) and a temperature range of 20.1–31.0 °C (average 21.6 °C) where tissue sections were not exposed to direct light.TM FLEX detection (High pH) on the Autostainer Link 48 (Agilent). Immunostaining for pan-cytokeratin (pan-CK) was assessed as a control using an antihuman Cytokeratin, clone AE1/AE3, ready to use . A representative section from each block was stained for hematoxylin and eosin (H&E) on day 0 of each experiment. All stained tissue sections were scanned on an Aperio ScanScope AT Slide Imager  at ×40 magnification, and images viewed on Aperio ImageScope (v12.3.2)  and humidity levels were measured using a Lockdown Hygrometer . Sections stored within the incubator were removed at appropriate time points for IHC staining alongside control sections stored in normal archival conditions . Repeat experiments of environmental effect on antigen degradation involved the change of these parameters individually. Experiments exploring the use of desiccant to protect against chamber conditions involved comparing antigen expression in sections placed within the chamber in a closed box, sealed in a protective bag  with desiccant  and a humidity indicator card ), to sections placed within the chamber without additional protection, as well as to sections stored under normal archival conditions.13C6, U-15N4-arginine, or U-13C6, U-15N2-lysine at the C-termini and unlabeled peptide standards were purchased from New England Peptide . Isotope labeled peptides were of greater than 99% and 95% isotopic and chemical purity, respectively; absolute concentration was determined by amino acid analysis.Synthetic, isotopically labeled PD-L1 peptide standards LQDAGVYR and AEVIWTSSDHQVLSGK containing U-PD-L1 was analyzed by targeted MS as described previously  with them/z values. Retention times were determined from prior analyses of synthetic peptide standards. The MS1 scan was collected at a resolution of 30,000, an automatic gain control (AGC) target value of 5e4, and a scan range from m/z 350 to 1000. MS1 data were recorded in profile mode. The MS1 scan was followed by targeted MS2 collision induced dissociation scans at a resolution of 30,000, an AGC target value of 5e4, 1.6 m/z isolation window, activation Q of 0.25 and an optimized collision energy for each target of 30%. MS2 data were recorded in profile mode. Parallel reaction monitoring transitions were extracted from raw datafiles and analyzed with Skyline . The mobile phase was initially 5% B and then programmed to 20% B over 18 min, to 35% B over 14 min and finally to 95% B over 5 min before recycling to starting composition. Targeted MS analysis was done by parallel reaction monitoring on the Lumos. The acquisition method consisted of a full scan selected ion monitoring event followed by targeted MS2 scans as triggered by a scheduled inclusion list, with a 5 min retention time window containing the precursor  Skyline . Peptidem/z 375 to 1500. MS1 data were recorded in profile mode. MS2 high-energy collision induced dissociation scans were acquired at an AGC target value of 1e4, a maximum injection time of 35 ms, and with an isolation window of 1.2 m/z. Tandem MS scans were acquired as centroided data. Peptide sequence identification from tandem mass spectra was done as described previously . The mobile phase was initially 6% B and then programmed to 27% B over 27 min, to 40% B over 40 min, and finally to 95% B over 8 min before recycling to starting composition. An MS1 scan was collected at a resolution of 120,000, an AGC target value of 4e5, a maximum injection time of 50 ms, and a scan range from eviously , except eviously . Peptidet test as appropriate. Relationships between variables were assessed using Pearson’s correlation and, if appropriate, linear regression. Analysis of the effect of incubation conditions on methionine oxidized peptides compared the baseline condition and day 28 accelerated degradation condition and used peptide count data for peptides with at least ten spectral counts using Fisher’s exact test (two-sided). The Wilcoxon Signed Rank test was used to determine significance in the number of oxidized peptides between the baseline and day 28 conditions. Analyses of spectral counts were performed using R software for statistical computing (version 3.4.3) and all significances were taken as p < 0.05.Statistical analysis was performed using IBM SPSS statistics software, version 25 . Comparisons of multiple groups over time were performed using repeated measures ANOVA with Bonferroni correction and Tukey’s post-hoc analysis. Comparisons of two groups at a single point were performed using independent samples or paired Student’s p < 0.001), but both clones showed significant loss of PD-L1 expression over time as expected . Examples are shown in Fig. p < 0.05). Clinically relevant loss of CPS (from ≥1% to <1%) was seen by 4.5 months for both clones  with further loss by 24 months .To confirm the PD-L1 immunoreactivity loss in stored tissue sections that has been reported in literature could be reproduced in our laboratory, TMA sections containing 35 gastric carcinomas stored in archive over 24 months under normal ambient conditions were stained for PD-L1 using E1L3N and SP142 clones. Positive pixel count scoring (positivity) demonstrated that E1L3N detected PD-L1 with a greater sensitivity than SP142  and strongly positive (TPS ≥ 50%) cases when assessed with the 22C3 , 28-8 , and E1L3N  clones, but fewer positive and strongly positive cases for SP142 . Loss of PD-L1 TPS in NSCLC sections to levels below these prescribing guideline cutoffs occurred for all clones at varying time points, with over half of cases changing from diagnostically positive to diagnostically negative by day 19 for 22C3 (TPS ≥ 1% and ≥50%), day 9 for 28-8 (TPS ≥ 1% and ≥50%), and day 9 for positive and day 19 for strongly positive for E1L3N  p < 0.001; CK in placenta, 0.626 vs 0.259 (100% vs 41%); p = 0.05, tonsil 0.319 vs 0.219 (100% vs 69%); p < 0.05). Control slides kept at normal ambient conditions had no significant loss of either PD-L1 or pan-CK expression by day 28 for both placenta and tonsil tissue.In order to further understand the relative contribution of major environmental conditions on tissue immunoreactivity, the effect of oxygen, humidity, and temperature on PD-L1 (E1L3N) and pan-CK (AE1/AE3) IHC expression was assessed on tonsil and placenta tissue sections stored in the acceleration chamber set to baseline conditions of 100% oxygen, 37 °C, and 80% humidity over 28 days. By day 28 significant loss of PD-L1 and pan-CK positivity was seen in both tissues (average positivity day 0 vs day 28: PD-L1 in placenta 0.528 vs 0.088 (100% vs 17%); p = 0.174) or pan-CK in both placenta and tonsil tissue ; p = 0.284, tonsil 0.293 vs 0.247 (100% vs 84%); p = 0.423) with a significant reduction seen only for PD-L1 expression in tonsil by day 28 (average positivity 0.135 vs 0.07 (100% vs 52%); p < 0.05) though this was significantly less than the loss seen under 37 °C conditions ; p < 0.05), results summarized in Fig. Changing the temperature in the acceleration chamber had a significant impact on the rate of IHC signal loss. Increasing the temperature to 60 °C (in the context of elevated oxygen and humidity) resulted in extremely rapid loss of PD-L1 expression: . Conversely, decreasing the temperature to 20 °C reduced immunoreactivity loss, resulting in no statistically significant loss of PD-L1 in placenta tissue by day 28 ; p = 0.201) and required 28 weeks for a significant loss in tonsil to occur (0.183 vs 0.021 (100% vs 10%); p < 0.05). The rate of PD-L1 expression loss in reduced humidity conditions was slowed to the extent that both placenta and tonsil tissue demonstrated loss by 39 weeks at 45% humidity similar to, or less than, 1 week at 80% humidity (average positivity for placenta 0.41 vs 0.17 (64% vs 27%); p = 0.13, and tonsil 0.018 vs 0.019 (10% vs 10%); p = 0.93). Average positivity of PD-L1 in placenta and tonsil at 28 days under 45% and 80% humidity are shown in Fig. Reducing humidity had a significant impact on the rate of IHC signal loss. Decreasing humidity to 45% (in the context of elevated oxygen and temperature) resulted in no significant loss of PD-L1 expression in placenta at 39 weeks (0.639 vs 0.408 (100% vs 64%); p = 0.918; tonsil 0.0079 vs 0.016 (7% vs 14%); p = 0.937; pan-CK, placenta 0.43 vs 0.45 (68% vs 71%) p = 0.918; tonsil 0.21 vs 0.22 (61% vs 64%); p = 0.937). Results shown in Supplementary Fig. Reducing oxygen levels in the incubation chamber to 20% (in the context of elevated temperature and humidity) had no significant effect on the rate or total quantity of either PD-L1 or pan-CK expression in either placenta or tonsil tissue ; Because humidity was determined to be a major factor in loss of tissue immunoreactivity, the effect of storing slides with or without desiccant in the acceleration chamber set to baseline conditions of 100% oxygen, 37 °C and 80% humidity over 28 days was examined. This was accomplished by measuring the expression of PD-L1 (E1L3N) and pan-CK IHC using positive pixel count scoring (positivity) on placenta and tonsil tissue sections stored over 28 days.p = 0.083, tonsil 0.088 vs 0.073 (100% vs 83%); p = 0.555, pan-CK in placenta, 0.74 vs 0.72 (100% vs 97%); p = 0.311, tonsil 0.33 vs 0.30 (100% vs 91%); p = 0.185).Upon removal of slides from the sealed container stored within the acceleration chamber, the humidity level was recorded as <30% using the enclosed indicator card. Slides stored with desiccant showed no significant loss of either PD-L1 or pan-CK expression in any tissue at day 28 (Average positivity day 0 vs day 28: PD-L1 in placenta 0.57 vs 0.53 (100% vs 93%); Qualitative assessment of sections showed slides stored with desiccant demonstrate expression loss similar to sections stored under normal atmospheric conditions, with appreciable loss of PD-L1 expression in sections stored without desiccant Figs.  and  5. Tonsil tissue stained for PD-L1 with E1L3N stored in the acceleration chamber with conditions of 100% oxygen, 80% humidity and 37 °C over 28 days was assessed for expression loss difference between crypt epithelium cells and ICs associated with germinal centers. Day 0 sections showed strong staining within the crypt epithelium, and weaker staining within the geminal centers, in keeping with known PD-L1 expression variation between these cell types . Loss isTo test the hypothesis that peptide oxidation is a major contributor to loss of PD-L1 immunoreactivity in stored tissues, targeted MS analyses of PD-L1 peptides were performed to measure PD-L1 protein abundance in specimens stored in normal atmospheric conditions or within the acceleration chamber with conditions of 100% oxygen, 37 °C, and 80% humidity. Two PD-L1 peptides were analyzed, LQDAGVYR and AEVIWTSSDHQVLSGK, both of which are in the extracellular domain of PD-L1. Whereas LQDAGVYR contains no easily oxidizable residues, AEVIWTSSDHQVLSGK contains an oxidizable tryptophan (W) and lies within the recognition sequence for the 22C3 antibody . It has r2 = 0.744, p = 0.001)  Fig. . MS anal01) Fig. .Fig. 6CoOxidation of PD-L1 tryptophan residue in AEVIWTSSDHQVLSGK would occur within the 22C3 recognition sequence and might affect antibody binding. Hydrogen peroxide treatment of the AEVIWTSSDHQVLSGK labeled standard yielded the kynurenine form AEVIW[+4]TSSDHQVLSGK as the predominant product. In MS analyses of the 28 day incubated samples, we detected the AEVIW[+4]TSSDHQVLSGK labeled standard, but not any oxidized peptide from the NSCLC samples . The distributions for ambient stored placenta and tonsil were similar to the 28 day wet oxidized NSCLC from the acceleration chamber . Although these global data indicate the presence of statistically significant oxidation, the distributions clearly indicate a modest overall degree of oxidation that could not account for the near-complete loss of PD-L1 IHC staining observed.To further test the hypothesis that the acceleration chamber produced oxidation of proteins in FFPE sections that interferes with immunoreactivity by IHC, we performed global MS analyses of four samples each of the baseline and day 28 samples from the acceleration chamber experiment, as well as two samples each from placenta and tonsil stored as unstained sections under ambient conditions for 2 years. We extracted data for a set of 274 methionine-containing peptides that were found in all samples in both reduced and oxidized forms and compared abundances as the log2 ratio of spectral counts for both forms (Fig. The loss of immunostaining in archived FFPE tissue is a well-known but poorly understood phenomenon . The rolThe loss of antigen immunoreactivity has been observed to vary between different antigens within and between tissue types, with no obviously consistent factors that may help predict the most sensitive antigens. Both polyclonal and monoclonal antibodies may be affected, and loss occurs in IHC assays that target the nucleus, cytoplasm, or membranes of cells. Specific IHC signal loss can vary between studies depending on factors such as fixation and the specific antibodies used , 37–39.Mechanisms of antigen degradation have been explored previously, with a variety of potential factors thought to influence the loss, including: preanalytical variables, oxidation, humidity, and high temperature , 40, 41.The role of oxidation in antigen degradation has been suggested previously , 40, andPrevious work suggested that humidity or the fixation process can have an effect on antigen degradation , 42–44. The crosslinking process increases accessibility to some antigens and renders others inaccessible through masking of epitopes , 50. TheTissue processing and antigen retrieval prior to IHC involves dehydration and a rehydration of tissue, which has been demonstrated to be a crucial step in achieving successful IHC assay outcomes , 57. IntThis study has limitations. While over a thousand tissue sections were included in the analysis, sample sizes were small for some conditions, perhaps accounting for nonstatistically significant trends in certain experiments. Positivity as defined by pixel counting was complemented by TPS/CPS scores in tumors to give clinical relevance, but the equivalent is not possible in placenta and tonsil; therefore, although significant loss can be quantitatively demonstrated with good reproducibility (Supplementary Figs. To our knowledge, this is the first study that has systematically used quantitative MS to characterize the impact of tissue storage using a model system under controlled conditions. We demonstrate that PD-L1 expression assessed by IHC with different antibody clones undergoes signal loss over time, and that this loss is largely accelerated by humidity and high temperature, rather than by environmental factors favoring oxidation. The use of an acceleration process to mimic the natural loss of antigenicity that occurs in naturally stored FFPE tissue sections may provide a platform by which novel biomarker robustness can be evaluated early in development. MS is a powerful technique that overcomes the limitations of studying protein expression in stored tissues sections by IHC alone. Moreover, MS may be ideally suited to analyze archival FFPE specimens and conduct hypothesis testing in regards to the abundance of protein drug targets with associated therapeutic outcomes.Supplementary figures S1–S9 and supplementary tables S1–S2


Digital assessment is becoming more and more popular within medical education. To analyse the dimensions of this digital trend, we investigated how exam questions (items) are created and designed for use in digital medical assessments in Germany. Thus, we want to explore whether different types of media are used for item creation and if a digital trend in medical assessment can be observed.In a cross-sectional descriptive study, we examined data of 30 German medical faculties stored within a common assessment platform. More precise, 23,008 exams which contained 847,137 items were analysed concerning the exam type  and their respective media content . Out of these, 5252 electronic exams with 12,214 questions were evaluated. The media types per individual question were quantified.The amount of computer- and tablet-based exams were rapidly increasing from 2012 until 2018. Computer- and tablet-based written exams showed with 45 and 66% a higher percentage of exams containing media in comparison to paper-based exams (33%). Analysis on the level of individual questions showed that 90.8% of questions had one single picture. The remaining questions contained either more than one picture (2.9%), video (2.7%), audio (0.2%) or 3.3% of questions had picture as well as video added. The main question types used for items with one picture are TypeA (54%) and Long_Menu (31%). In contrast, questions with video content contain only 11% TypeA questions, whereas Long_Menu is represented by 66%. Nearly all questions containing both picture and video are Long_Menu questions.It can be stated that digital assessment formats are indeed on the raise. Moreover, our data indicates that electronic assessments formats have easier options to embed media items and thus show a higher frequency of media addition. We even identified the usage of different media types in the same question and this innovative item design could be a useful feature for the creation of medical assessments. Moreover, the choice of media type seems to depend on the respective question type. Artificial intelligence, big data, university 4.0, digital transformation and digitalisation are current trends and buzzwords highly discussed in the world of health care and higher education –3. EspecDigitalisation encompasses the emergence of novel tools and applications which offer many new opportunities within the educational sector, especially concerning new assessment formats and content of exams . For thiThe potential addition of media content to questions is the benefit of E-Assessment. In the context of medical education, a study area with the most performed electronic exams , electroThe usage of digital tools on mobile devices allows a layered and dynamic item design, and thus the medical educator has become a designer of items in assessments . The conThe basis for our data evaluation is the assessment platform ItemManagementSystem (IMS) of the Umbrella Consortium for Assessment Networks (UCAN) , 20, wheFor the investigation of the media usage in exam questions, 17,756 paper-based exams were disregarded because paper-based exams offer less possibilities for media addition compared to electronic exams. Thus, the remaining 5252 computer- and tablet-based exams with 205,988 questions were further analysed. For tablet-based exams we distinguish between tablet-based written assessments (tEXAM) and Objective Structured Clinical Examinations (OSCE) evaluated by the examiners via tablet (tOSCE). tOSCE was introduced in 2013 and tEXAM in 2015.TypeA questions are multiple-choice questions with one correct answer.Pick-N questions are also multiple-choice questions, but with more than one correct possible answer.Kprime questions are basically yes or no questions where each possible answer must be marked as true or not true.Freetext questions have a textbox where any desired answer can be given.Long_Menu question have usually a long answer list  to choose from and thus are comparable to freetext questions where the knowledge of the right answer is presumed.OSCE is an assessment format for skill testing in written or tablet-based form that was first described by Harden et al. , 22. In First, the different media types per single question were quantified and second the preferred question type for each media type determined. We categorised the questions into six different classes: TypeA, Pick-N, Kprime, Freetext, Long_Menu and OSCE:The evaluation of the compiled data occurred through the software packages Microsoft Excel . With the Excel software mean values and standard deviations were determined. The dependency of question type and media type was analysed with R  using Pearson’s Chi-squared test.Figure The average percentage of exams containing media with reference to all exams of the respective exams type is illustrated in Fig. In the investigations what kind of media types are used in electronic exams, it is not surprising that pictures are still the main media type used in combination with text Fig. . AdditioTo analyse the used media type in E-Assessment, 5252 electronic exams were evaluated with 2065 exams that contained either picture, video or audio as media types. These exams contained 12,214 questions that were further assessed individually regarding their added media types. In accordance with the above-mentioned data, the evaluation of questions showed that pictures are indeed the main media type in combination with the question text .We investigated whether a particular media type is favoured for a special question type. Our findings show that 54% of the questions containing one picture can be assigned to single answer multiple choice questions (TypeA) (Fig. The current study shows the digital trend towards electronic exams in German medical schools. The quantity of electronic assessment methods, such as computer- and tablet-based exams has grown strongly until the end of 2018 according to our analysis. The frequent usage of picture items could be demonstrated with our data since pictures still represent the main media type used in medical assessments. Our data shows that other media types such as audio and video are quite rarely used within medical education in Germany. Perhaps these media types are more advantageous for other areas of studies. Within the musicology, parts of musical works could be played and learning foreign languages native speakers could be listened to which is already an element of learning management systems –25 and pIn general, good quality questions used for exams are costly  and one Unfortunately, the authors did not find any references regarding nationwide studies to determine a change in media usage after implementation of digital exams. However, there is a trend in the use of digital assessment formats worldwide –32. As eThere are some limitations to our study. First, we only analysed the data of one specific database which depends on the accurate entering of the data by its users. Second, we have only focused on medicine as a subject of study and only considered German medical faculties. Third, our exam exclusion criteria may have resulted in exclusion of potentially relevant exams or questions. Moreover, the number of some question type and media type combination in our analysis might be rather low for less frequently used question types. Nonetheless, this study is the first attempt to compare and analyse the used media types for medical exam questions in different exam types.Further research could incorporate the analysis whether the use of media in exam questions is beneficial for the quality of the assessments and thus the quality of medical study. For that reason, statistical data like the difficulty, discriminatory power and reliability of exam questions with and without media should be collected. An automatic upload of statistical values does currently not take place in the examined assessment platform, but is planned for the current year, which is why a statistical analysis of exam questions would be feasible in the future and is aimed at. Furthermore, it should also be explored to what extent different medical competencies and knowledge can be tested depending on the chosen media and question type. Hurtubise and colleagues discussed the usage of video clips linked to different competency domains . In GermIn the age of digitalisation, a digital trend is occurring in higher education in Germany. In our study we analysed how this digital change is affecting assessments in medical education. Interestingly, an increased number of media additions like picture, video and audio can be observed in electronic exams and even media combinations of text, picture and video are utilised for the creation of exam questions. So far, the usage of different media types in the same question is quite rare, but modern digital technologies together with the collective exchange of items in a common item-pool and a broad range of available assessment tools will facilitate these innovative digital assessment formats in the future. In addition, our study demonstrates that some media types are more used for a specific question type than others. Thus, one could speculate that our data shows that specific media types are more suitable for some question types than for others.


This review is focused on the recent developments of nanocomposite materials that combine a thermoplastic matrix with different forms of graphene or graphene oxide nanofillers. In all cases, the manufacturing method of the composite materials has been melt-processing, in particular, twin-screw extrusion, which can then be followed by injection molding. The advantages of this processing route with respect to other alternative methods will be highlighted. The results point to an increasing interest in biodegradable matrices such as polylactic acid (PLA) and graphene oxide or reduced graphene oxide, rather than graphene. The reasons for this will also be discussed. The fabrication of nanocomposites by the incorporation of nanofillers into polymer matrices originates materials with new functional properties while the excellent processing and manufacturing abilities are retained due to the flexibility in polymer matrices . In receIndustrial thermoplastic polymers and composites are usually melt-processed by extrusion followed by injection molding. The extrusion step combines the application of high temperature and high specific mechanical energy in a short time interval. The optimization of extrusion parameters plays a fundamental role on the final properties of the material. Extrusion elements configuration and parameters such as residence time, screw speed, temperature and energy are even more critical when nanocomposites are concerned ,11. The The original definition of graphene  is that Solvent processing. The use of conventional solvents causes serious health and environmental problems which prevent the large-scale usage of these procedures.In situ polymerization. This is an effective method to obtain small quantities of materials for laboratory research applications but not for industrial scale.Melt blending. Several thermoplastic matrices have been modified by melt blending addition of different forms of graphene oxide (GO) and reduced graphene oxide (rGO) nanofillers. However, this process might cause defects in the GO structure, from wrinkles and rolling, to modification of the initial aspect ratio.Methods for achieving the dispersion of graphene in polymer matrices  usually Methods using a solvent need the previous modification of graphene surface in order to disperse it. In situ polymerization can be more efficient. However, both methods are not readily scaled to industrial processing. In contrast with solvent dispersion and in-situ polymerization processes, melt processing, that is, the direct dispersion of graphene nanophases into molten polymer is a cleaner process that can be readily scaled up for industrial applications. However, in order to achieve uniform graphene dispersion into highly viscous polymers, it is still necessary to modify the surface of graphene flakes. Although this modification can be made by chemical functionalization, with the formation of new covalent bonds , it can Melt-processing is usually performed by twin-screw extrusion, which is an environmentally-friendly method immediately applicable to the industrial scale manufacturing of final parts. The versatility of this process allows tailoring the extrusion parameters for each material and application. This method is widely used for the processing of nanocomposites, since the mixing action is based on an elongational flow. Shear and elongation stresses are as the material is transported through the space between screws  .2, the order of magnitude of the force necessary to exfoliate the graphite is about 300 nN/mm2 [Another advantage of extrusion could be the alignment of planar nanostructures such as those of graphene nanomaterials in one preferential direction. Kim et al.  have dis0 nN/mm2 ,26,27, wResearch interest in nanocomposite materials containing graphene has been present for more than a decade , as it b2 hybridization. As it is depicted in As it is mentioned above, there are several kinds of carbon nanofiller that are commonly called graphene. The basic structure of the different graphenic nanomaterials is graphene . This na3 hybrid carbon atoms with functional groups such as hydroxyl and epoxy groups in the surface and carboxylic groups at the edges [One of the most widely used graphene derivatives is graphene oxide. The structure of this nanomaterial consists in graphene sheets with stoppages of sphe edges ,40,41. The edges ,43. Othehe edges ,45,46. IIn this section we have summarized the recent publications on thermoplastic matrix composites modified by addition of graphene-derived nanofillers, modified graphene nanofillers or blends with other nanomaterials. These new nanocomposites have been obtained by extrusion, in most cases twin-screw extrusion, which can be followed by injection molding.The effect of graphene nanoplatelets (GnP) size on PP nanocomposites processed by twin-screw extrusion and injection molding has been studied  to find Recently, the combination of several nanofillers has demonstrated a useful strategy to obtain new nanocomposites with special properties. Synergy between graphene (G) and synthetic graphite (SG) was studied by Altay et al. , when adIn any case, agglomeration is an impasse in the development of PP-based nanocomposites . During Although PP and PLA (see below) are the most widely studied matrices for graphene nanocomposites, many other thermoplastic materials have also been modified to obtain graphene nanocomposites  21,64,6,92,93,94Polyethylene (PE) and different types of polyethylene-derived materials such as linear low-density polyethylene (LLDPE), high density polyethylene (HDPE), or polyfluorinated ethylene propylene and polyethylene naphthalate (PEN) have been studied as matrices ,67,68,69Polyamides (PA) are another commonly used base polymer for the processing of graphene containing nanocomposites ,75,76,772 processed by using twin-screw extrusion [Other widely used thermoplastics include polystyrene (PS), thermoplastic polyurethane (TPU) or poly (methylmethacrylate) (PMMA) matrices ,78,79. Ixtrusion  have beextrusion  to reducxtrusion . ABS  , PPS  of PLA has been explored ,109,110.Biodegradable nanocomposites have been obtained by different approaches, such as thermal reduction of GO during processing of PLA-PBS (polybutylene succinate)-rGO nanocomposites , and theco-3-hydroxyvalerate)), the selected fillers are a combination of rGO and ZnO nanoparticles [In the case of the biodegradable and biocompatible plastics, different mixtures of graphene and other materials are used as fillers to preserve the sustainability of the composites. Manufacturing of biodegradable plastics can reduce the environmental impact and favor renewable resources. However, some properties of biodegradable polymers cannot often be comparable with petroleum-based plastics and the addition of fillers may enhance the physical, chemical and mechanical properties ,115. Bioarticles , to mainThe present work is an overview on very recent developments in the field of graphene-thermoplastic nanocomposites melt processed by extrusion methods. A wide range of matrices have been studied, from commodity polymers to high resistant engineering polymers. Special attention is being paid at the development of biodegradable and biocompatible nanocomposites.Extrusion is readily scalable to industrial manufacturing of final products; however, in most cases, obtaining homogeneous graphene dispersions within the polymer matrix is not a simple task. Segregation and agglomeration of the nanophases are responsible for the lack of improvement in the thermal, mechanical and electrical properties with respect to the unmodified matrix materials.The results of this review denote that the most widely used techniques to improve the dispersion of graphenic nanofillers are sonication, surface modification and the application of a coating layer of graphene on the polymer surface. However, another promising technique that is beginning to yield positive results is the use of ionic liquids, which should be explored more thoroughly. Other strategies are focused on the modification of the extrusion process, the use of masterbatches and the combination of graphene with other materials as hybrid nanophases.The main factors that affect the characteristics and attributes of these nanocomposites are the nanofiller size and concentration. It has been stated that larger sizes of nanofillers result in a reduction of the mechanical properties but an increase of other functional properties, such as electrical conductivity. However, the effect of graphene concentration is not so well defined, and more studies are necessary.A relevant field, which has received limited consideration until the present moment, is that of the influence of the additives on the deformation and fracture modes and on the resistance to degradation of the final materials.Finally, another line of work that is starting to receive attention is the tribological performance of extruded nanocomposites as a function of extrusion parameters, polymer structure, nanofiller orientation and surface modification.


Graphene as the 2D material with extraordinary properties has attracted the interest of research communities to master the synthesis of this remarkable material at a large scale without sacrificing the quality. Although Top-Down and Bottom-Up approaches produce graphene of different quality, chemical vapour deposition (CVD) stands as the most promising technique. This review details the leading CVD methods for graphene growth, including hot-wall, cold-wall and plasma-enhanced CVD. The role of process conditions and growth substrates on the nucleation and growth of graphene film are thoroughly discussed. The essential characterisation techniques in the study of CVD-grown graphene are reported, highlighting the characteristics of a sample which can be extracted from those techniques. This review also offers a brief overview of the applications to which CVD-grown graphene is well-suited, drawing particular attention to its potential in the sectors of energy and electronic devices. The gases are then passed through the reactor at a predefined ratio. The partial pressures of the hydrocarbon and the hydrogen can be controlled by changing the reactor pressure or by adding an inert diluent gas such as argon (Ar) or nitrogen (N). After the reaction, the furnace is typically switched off and allowed to cool under a gas flow  until the reactor reaches room temperature. This step is essential in order to avoid aggregation of the deposited carbon, which may result in multi-layer graphene or bulk graphite. It thus contributes to achieving a continuous graphene monolayer on the surface of the metal. In the case of CVD graphene deposition, a metal substrate such as copper (Cu) is placed into a furnace tube made from quartz and heated under a flow of hydrogen at low vacuum or atmospheric pressure. The elevated temperature anneals the substrate which leads to enlargement of its grain size  , as well4 is catalytically decomposed and where the carbon atoms are adsorbed to form graphene film, while, in the high carbon solubility substrate, carbon atoms are diffused into the bulk, and the graphene growth step happens during the cooling step when carbon precipitates to the surface. Fast cooling is thus recommended to avoid the formation of multi-layer graphene. In surface processes (step 6), catalytic decomposition of reactive species occurs in addition to surface migration to attachment sites and other heterogeneous reactions. By-products are then desorbed from the substrate (step 7) after film growth. Finally, by-products are diffused through the boundary layer to the main gas stream (step 8) to be carried away by the force of convection to the exhaust system (step 9) . . 107]. I in 10 s . Furthers growth .2 ..4 ratio In conclusion, SEM has been extensively used as a powerful tool for graphene characterisation, as numerous amounts of information can be extracted from SEM images. Optimising the SEM operating conditions such as acceleration energy, working distance and determining the right detector enables efficient visualisation of surface features in the grown graphene via CVD methods.Atomic force microscopy (AFM) is an extensively used microscopic characterisation technique for graphene characterisation. 3D images obtained using AFM enables the measurement of the thickness of graphene films and the lateral dimensions, which, in turn, allows for accurately determining the number of layers present, thermal conductivity, electric and optical properties of graphene-based products ,195.The necessary component of the AFM instrument is a nanometre-sharp tip created using micro-fabricating technology, which is attached to an adjustable cantilever. The cantilever serves as the transducer to sense the sharp tip on a graphene sample, while a tip acts as a probe that scans over a sample line-by-line . The intThe initial step in setting up the AFM device for spectroscopic and imaging analysis involves mounting the probe in the cantilever, which is typically done with forceps. The removal of the probe for storage is done with vacuum tweezers. Correct mounting is a critical requirement for accurate measurements and ideal laser alignment . The intThe determination of the number of layers is the most common application of the AFM. The procedure typically involves scanning the edges of graphene sheets at the places where the stacked layers form steps . In mostTo date, several AFM-based electrical characterisation techniques are available, a detailed review of which is given in a study by Hussain et al. . ContactThe AFM in nanoindentation mode serves as an effective means of testing the mechanical properties of graphene materials. The procedure involves measuring the intensity of subtle interactions between the sample surface and the probe during the loading stage and the unloading stage and recording them on a force–distance curve. The analysis of such curves enables the determination of numerous characteristics and mechanical properties of graphene-based thin-films, including the hardness, stiffness and Young’s modulus ,201. TheOther morphological features in graphene film can be characterised using AFM such as graphene ripples. Chaitoglou and Bertran have studied the effect of growth temperature on CVD grown graphene where ripples were observed on the surface. AFM was utilized to obtain hight measurements for the ripples; the temperature showed no significant effect on the geometry of the ripples. However, the measured step height gave a value of 5 nm, which is in agreement with the calculated depth value using the model of Mullins  78]. . 78]. 2C. The thickness of the Mo2C hexagonal domains increased with growth time, giving heights of approximately 40, 140 and 190 nm for growth times 20, 60 and 90 min, respectively  is a powerful spectroscopy technique that provides valuable information about the structure of a material at the atomic level . It was The TEM is one of the most versatile and reliable methods for studying graphene edges. The low atomic number of graphene implies decreased scattering of the electron beam, which, in turn, allows for sharper TEM images. STEM and HR-TEM imaging are practical tools for the following objectives: determining the number of layers, studying the atomic structure and characterising the electronic properties of both graphene and graphene doped with heteroatoms . In addiTEM is also used to rotational misalignment between graphene layers using Moire patterns while Fourier transform of the electron diffraction pattern provides information on the nature of the graphene film as demonstrated by Nayak  209]. I. I209]. 2 only, but sp3 hybridization might appear locally.TEM is also considered as an excellent tool to study the structural defects in graphene which have a strong influence on the properties of the produced film that will limit their implementation. Point defects cause undesired electron scattering that drastically reduces electrical conductivity. Bonds around defects become weaker, which decrease the mechanical strength and affect the thermal conductivity. Therefore, understanding of the formation and the nature of those defects in graphene structure is essential in order to tailor the growth conditions and substrate properties to get high purity graphene with no defects. A review on the structural point and line defects in graphene were reported by Banhart et al. as they demonstrated the theoretical defect formation (intrinsic and extrinsic) as well as the utilisation of TEM as an imaging technique for those defects . DefectsSimilarly, adatoms defects can occur by a foreign atom (non-carbon) which affect the graphene properties; the effect will primarily depend on the interaction bond between the foreign adatom and the nearest carbon atoms . SubstitGraphene can also be considered an ideal TEM support film. CVD graphene provides supports of extreme physical stability, periodic structure, single-atom thickness and ballistic electrical conductivity . The ult2/Vs), outstanding flexibility, high transparency in near-infrared and visible light, graphene and graphene-based materials can be applied in a wide variety of applications in electronics, energy, biomedicine and environmental engineering [Due to its unique set of material properties, including exceptional carrier mobility , hence, intensive research is ongoing in order to induce a bandgap in graphene to boost its use digital electronics applications. However, the range of applications for graphene in electronics extends far beyond just digital systems. Here, we briefly review the implementation of graphene in light emitting diodes (LEDs) and nano electromechanical systems (NEMS), highlighting how this helps to develop novel sensing devices and wearable electronics for health monitoring. Since the invention in 1962, LEDs have been used almost universally in a considerable number of different electronic devices. Generally, LED voltage is relatively high because it is required to be equal to or higher than the bandgap energy. These semiconductor light sources necessitate electrodes with high electrical conductivity and high transmittance to enhance their optical and electrical characteristics. However, transmittance and conductivity are mutually contradictory properties, which leads to significant difficulties for simultaneously satisfying both requirements . A set o−1. Although the transmittance of graphene (about 90%) satisfies the requirement, the sheet resistance of CVD-grown graphene exceeds the acceptable level for industrial applications. Several doping methods that employ metal hybrid composites have been proposed to improve the electrical and structural properties of graphene-based materials [Today, indium tin oxide (ITO) remains the most widely utilized transparent conductive electrode (TCE) in optoelectronic devices. However, the scarcity of indium inevitably leads to a constant increase in the cost of ITO, severely limiting its use for low-cost, sustainable and transparent thin-film devices . The synaterials .2+) as dopants from the upper electrode into monolayer graphene films grown by the CVD method through conducting paths formed in aluminium nitride  under the application of an electric field. The authors reported the studied material retained a very high optical transmittance of about 95% after the doping process, while the work function of graphene increased from 4.36 eV to 5.0 eV as a result of p-type doping effects of Ni atoms. The LED with the Ni-doped monolayer graphene demonstrated superior light output power characteristics compared to both LEDs with a 100-nm-thick ITO electrode and LED with undoped graphene without any significant losses in the current–voltage characteristics [A study by Chae et al. describes a novel electric-field-induced doping treatment, which can effectively reduce the sheet resistance of CVD-grown monolayer graphene while retaining its high transmittance. The treatment involves the injection of metallic ions  and industrial mass synthesis makes this metal an especially attractive candidate for use as TCE in optoelectronic devices. However, the application of Cu nanowires in LEDs is severely limited by the low resistance contact and poor stability against oxidation. Huang et al. proposed a CVD method for fast gas-phase encapsulation of the graphene shell layer on Cu nanowire networks to effectively improve the optoelectronic performances of TCEs and enhance oxidation resistance. The authors reported the successful fabrication of fully transparent GaN-based LED chips on a wafer using the Cu graphene nanowire TCEs. The central emission peak of this device is located at 437 nm in the blue region without any additional peaks associated with impurities, which indicates the excellent light transmission and high stability .One of the promising applications of graphene-based material in the field of LED devices is their use as a replacement for conventional sapphire substrates for growing semiconductor materials for LEDs. A study by Li et al. reports the successful growth of an InGaN/GaN blue LED on wet-transferred multilayer graphene. The use of graphene as a substrate allows for releasing the stress during the growth of indium gallium nitride, which leads to a noticeable decrease in the number of cracks. The authors reported that the blue InGaN/GaN multiple quantum wells LEDs grown on graphene exhibit a higher output power and a slightly reduced crystalline quality of GaN compared to that of the LEDs grown on a conventional sapphire substrate .Despite the outstanding set of desirable material properties, the implementation in stretchable electronic applications has been severely inhibited by the tendency of graphene-based materials to crack at small strains . Defects−1 [−1 at room temperature [2 substrate using lithographic techniques, and metal contacts were made of Cr/Au (5/100 nm). The resonant frequency and QR were measured in vacuum condition in a chamber, followed by a set of measurements while a mixture of hydrogen and argon were injected, thus changing the pressure in the chamber. The results showed the change in adsorbed molecules (mass in zeptogram) with the pressure change which proves the efficiency of graphene NEMS-resonators for spectrometry applications for light mass molecules. Another interesting implementation of graphene based NEMS-resonator is the research conducted by Andelic et al. for the detection of chemical warfare agents (CWAs) where they applied mathematical methods and frequency shift method to detect an attached CWA molecule [Nano electromechanical systems (NEMS) are devices that integrate electrical and mechanical functionality. Graphene provides a high surface to volume ratio, which leads to high sensitivity in the NEMS while maintaining a stable mechanical properties. Moreover, graphene’s geometry makes it suitable for standard lithography while its stiff yet flexible material and its electrical conductivity enable its integration in electrical transducers . In anot−1 . On the perature . Their rperature . Graphenperature . The senmolecule . Fan et molecule . 2 [2 sensing [2 concentration and the sensor response, while the performance of the graphene-based sensor was evaluated in dry and humid conditions (0% and 50% humid environment) showing a less than 5% degradation in humidity, which is much lower than those of metal oxide gas sensors under the same conditions. Some excellent dedicated reviews about graphene in NEMS have been reported, discussing both NEMS and MEMS applications [Another interesting property in graphene is that it exhibits an excellent piezoresistive effect, which makes it ideal for piezoresistive pressure and strain sensors. In 2013, Smith et al. developed a highly sensitive pressure sensor using monolayer CVD grown graphene membranes that were suspended over the patterns on SiO2 . The sen2 . The res2 . Both se sensing . They prications ,239,240.As aforementioned, it was demonstrated that the implementation of CVD grown graphene in conventional NEMS structures where they still consist of transistor-like nanoelectronics shows superior performance and sensitivity . TraditiChen et al. reported the implementation of PECVD grown graphene nano walls (GNWs) in the fabrication of flexible electromechanical biosensor for real-time measurement of lactate . In the A combination of superior mechanical strength, flexibility, high optical transparency and superb carrier mobility makes graphene a great candidate to be used in energy applications. Here, we discuss three examples of using CVD grown graphene in this category, briefly reviewing the utilisation of graphene in solar cells, batteries and supercapacitors. 24 J of solar radiation per year, which is approximately 10,000 times the current global energy consumption. Thus, as solar energy has great potential in providing energy security, research is continuously tackling the main challenges in solar cells and photovoltaics (PVs) in order to reduce the cost and find novel approaches to increase their efficiency. The current market of conventional solar cells is dominated by crystalline silicon which is a well-established technology. Recent research reported Si-based solar cell with an efficiency of 26%, which is close to the theoretical limit of 29.1% [Solar energy is the most abundant sustainable form of energy on the planet. Earth receives 3 × 10of 29.1% . Howeverof 29.1% , CIGS [2of 29.1% , perovskof 29.1%  and CZTSof 29.1%  showing of 29.1% . Researcof 29.1% . Howeverof 29.1% .x dielectric layer which was deposited using PECVD with a designed lithography mask on the front surface of GaAS. The exposed area in the dielectric layer is considered as the active area for the graphene/GaAs solar cell, where the CVD graphene was transferred to the active GaAs area. The performance of the fabricated solar cell was tested using a solar simulator giving 18.5% power conversion efficiency (PCE) while drift–diffusion simulation was utilised and higher PCE was predicted with over 23.8% shedding the light to great potential of utilising graphene in PVs by investigating more dielectric materials [3) and antireflecting coating of PMMA were performed on the optimum graphene thickness in order to increase the PCE, reaching an efficiency of 9.18% [Since then, many studies have been made in order to increase efficiency by using an interfacial layer, doping and antireflective coating. Song et al. in 2015 reported a drastic increase in efficiency after using an optimal oxide interfacial layer, followed by a further increase to reach 12.4% after chemical doping followed by 15.6% efficiency by applying an antireflective coating, demonstrating a method for the combination of the three aforementioned techniques . Later, aterials . Furtheraterials . Graphenof 9.18% . Another type is the Perovskite solar cells, especially combining crystalline silicon in the design of the solar cell, is predicted to have high PCE using a hybrid of perovskite/silicon tandem devices. However, such a design requires the use of transparent conductive electrodes. Conventional transparent conductive oxides cannot be directly deposited on the top surface of the perovskite cell, requiring buffer layers leading to current losses. Promising alternatives are electrodes made of the silver nanowire (AgNW) as they have low sheet resistance, giving an efficiency of 17%; however, their optical transmission is 87%, which is far below that of graphene . Lang etOrganic solar cells (OSCs) have gained a worldwide interest due to their potential of having flexible and cost-effective solar cells. Organic solar cells consist of substrate, anode, hole transport layer, absorber, electron transport layer and a cathode. The anode layer is typically a transparent conductive layer made of indium tin oxide (ITO) for its electrical conductivity and optical transparency. However, ITO is brittle, making it unsuitable for the next generation of flexible devices. Additionally, since indium is a strategic material, alternatives are very desirable for the industry. Graphene has been proposed as the ideal replacement for ITO due to its properties. The efficiency of flexible OSCs is still low compared to other types of solar cells. However, efforts are ongoing to develop their efficiency further. Kim et al. investigated the use of CVD grown graphene as the transparent conductive electrode on flexible PET substrates . They sh2Sx, x = 4, 6 and 8) are highly soluble that they dissolve and shuttle through the electrolytes [Batteries are usually categorised as rechargeable, which mainly consists of lithium and non-rechargeable such as alkaline and zinc-based batteries. Briefly, the non-rechargeable contain two electrodes and an electrolyte where the charges flow in one direction and usually have a short lifespan. While rechargeable, it also consists of two electrodes, where the cathode is made of lithium-based metal oxide, and the anode is a porous carbon and having lithium ions flow in both directions (charging/discharging). The increasing demand of mobile electronic devices  requires high energy and power density storage systems that provide fast charging and stable performance. The traditional lithium-metal oxide batteries have many limitations, making them unable to meet those requirements . A suggetrolytes  and betwtrolytes .2S2/Li2S, which suppressed the shuttle effect. Therefore, this decreased the self-discharge compared to a conventional Li-S battery using a standard PP separator without sacrificing energy density. In another study, Assegie et al. reported an anode-free Li-metal battery consisting of a Cu current collector that is modified and coated with multilayer CVD grown graphene [Therefore, the need for a conductive material that is thin with negligible weight and yet flexible to be used as separator coating, CVD grown graphene film arise as the most promising candidate to enhance the power and energy density of Li-S batteries. Du et al. prepared a Li-S battery with polypropylene (PP) separator that is coated with two layers of CVD grown graphene . Enhancegraphene . They foMore studies showed the potential of using nanoscale silicon in anodes for lithium-ion batteries since they have high cycle stability and reversible capacity compared to bulk silicon . The siz−1 if the entire surface area is utilised [Supercapacitors have attracted enormous interest in research communities due to their outstanding potential in terms of high power density, high charge/discharge rates and long lifespan performance. They are mainly divided into two categories based on the mechanism of their energy storage; (1) electric double-layer capacitors (EDLCs) and pseudo-capacitors . The EDLutilised ,274, but2So4 electrolyte, while Ag/AgCl was used as a reference anode. The supercapacitor performance showed an excellent charge/discharge cycling stability, and it sustained 84% of its initial capacitance after 1000 cycles. This revealed that graphene/Ta wire electrode is such a promising electrode material for high energy storage devices since it exhibited the highest capacitance of 345.5 F g−1. A recent study on reducing the equivalent series resistance (ESR) was reported by Kwon using optimised CVD grown graphene on Ni foil using different cooling rates  [−1). This was explained by the reduced ESR, thus providing a better interface, enabling more contact points between the electrode and the current collector. This shows advancement of the implementation of G-based electrodes in a supercapacitor for devices that require faster scan rates.Li et al. were the first to report the capacitance of graphene/tantalum (Ta) electrodes . They sy °C/min) . Graphen2. This was followed by more efforts indirectly use self-assembled and chemically modified graphene sheet to fabricate graphene-based fibres employing facile and spinning processes [2. The results showed remarkable porous structure (uniform pore distribution) and electrochemical performances in terms of discharge capacitance and cycling stability shedding light on its potential use as an electrode in supercapacitors. Later, the same research group , studied the implementation of their previously reported graphene fibre structure derived from CVD grown graphene into a flexible all-solid-state supercapacitor using gel electrolyte made [2 nanoparticles) structure worked as the active electrode and electron collectors. The areal capacitance of the graphene-based supercapacitor was reported to be 42.02 mF cm−2, which was considerably high compared to other fibre-based supercapacitors. In addition, the structure withstood mechanical bending and exhibited stable galvanic cycling as well as electrochemical performance.Another interesting implementation of 2D grown graphene films via CVD is their ability to be used in flexible solid-state (the electrolyte is non-liquid) supercapacitors. All-solid-state supercapacitors attracted the most attention due to their simple design, flexibility and efficiency in energy storage, enabling their use in wearable devices . Early srocesses ,279. Li rocesses . The novyte made . The gra2SO4) dropped cast to the supercapacitor design. The reported area specific capacitance of 5.0 µF cm−2 using the fabricated micro-supercapacitor was comparable to other reported supercapacitors based on CVD has grown graphene [More recent study was reported by Kim et al. in 2017, a novel roll-based patterning technology for CVD grown graphene was presented using highly durable Ni stamps to pattern the graphene at room temperature . The metgraphene ,284.An in-depth review was conducted on the synthesis of graphene using CVD methods  and the main characterisation techniques to study the characteristics of the produced graphene . A detailed review on the used growth substrates including the morphological effect and the nucleation and growth mechanism in solid and liquid substrates. Finally, an overview on graphene integration in applications, highlighting electronics and energy sectors in particular.In this review, the various factors affecting the quality of graphene film and its growth process were discussed, including the system total pressure, partial pressure of hydrogen and hydrocarbon species, growth temperature, source of power, etc. Knowledge of the effect of growth conditions and their correlation with the physical and chemical properties of the growth substrate is essential in creating a fundamental understanding of the process. In addition, it gives a powerful tool for tuning the growth parameters accordingly to achieve the desired graphene film, whether, monolayer, bilayer, few layer, or doped graphene which directly influence the electronic properties of the resulted graphene film. The progress in synthesis techniques and exploring wider range of parameters space in order to gain control over the deposition process to have the ability for large scale production while maintaining high quality graphene properties. Growth substrates were discussed in terms of the effect of pre-treatment methods and the surface morphology on the uniformity of the grown graphene film. Liquid substrates show the most potential to grow uniform, single crystalline monolayer graphene with no or a negligible amount of defects due to the absence of grain boundaries and other morphological defects when the growth starts on liquid state substrate. However, more investigations need to be done to explore other substrates such as low-melting point metals and precisely monitor the shrinkage effect of the substrate on the grown graphene.Overview of the essential characterisation techniques were discussed. Utilising Raman spectroscopy, scanning electron microscopy, atomic force microscopy and transmission electron microscopy reveals a tremendous amount of information about the characteristic properties of the produced graphene film as well as the defects nature and density. In addition, progress in the integration of CVD grown graphene in electronics and energy applications was discussed. Graphene’s role in the impressive performance enhancement of LEDs, NEMS, biosensors, solar cells, batteries and supercapacitors were highlighted. The window for graphene applications is wide open for limitless fields. However, CVD methods produce the best electronic quality for graphene, which makes it ideal for electronics and energy technologies in particular.Future prospects shall include novel designs for cost effective CVD systems that allow the continuous growth of high quality graphene on a large scale. Moreover, synthesising functionalized graphene and other 2D materials need to be researched as it could pave the way for a wider range of possibilities. Such novel materials can be employed and be very beneficial in enhancing the performance of electrochemical and electromechanical micro and nano devices. For example, their integration in ultra-sensitive biosensing for healthcare and medical sector as well as environmental sector  and evaluating the performance under harsh conditions are interesting fields that require further studies.


PREcise and Fast Method of Ambiguity Reinitialization/Resolution (PREFMAR) for L1 and L2 in GPS measurements. The method determines This paper presents a  Attempts to determine the total ambiguities in phase measurements in GPS observations date back to the late 1970s—i.e., GPS satellites were first placed in Earth orbits in 1978 . The firThe combination of L1-L2 frequencies of a GPS system is a special one, available in every multi-frequency GNSS receiver and probably the most effective double-frequency combination of GNSS data. A detailed description of mathematical functions used in the PREFMAR approach was investigated earlier for the use of L1-L5 or E1-E5a GPS/GALILEO data , but eacions see . In SectLet us make double-difference equations for two satellites, two measurement points and two frequencies: L1 and L2. For a very short baseline, the ionospheric and tropospheric delays have been ignored. Then, for the two frequencies, double-difference (DD) equations for carriers ; included .This means that for the frequency L1-L2, we should try to obtain code accuracy of less than 0.86 m, and then for short vectors we give only four sets of ambiguities—i.e., The dependence of the number of ambiguity sets on the accuracy of code measurements below 0.86 m using the PREFMAR method is shown in In In addition, in the case of reinitialization in time (t), we use the function values from the time before the disturbance in the reception of the continuity of phase signals—i.e., from time For the DD observation at time (t) during reinitialization, we can, therefore, write the following functions:The developed algorithms were tested on real L1-L2 observations, for a 10-min session with an interval of 1 s, and a baseline length of 3.6 km. The L1-L2 GPS measurements were performed with the Topcon HiperPro (M) receiver on 13rd August 2010, from 10:30:00 to 10:40:00 UTC, with reference to the OLST reference station. There were five GPS satellites available above the horizon: G09, G12, G15, G17 and G27, for which the following double-difference (DD) observations were created: G27-G09, G27-G12, G27-G15 and G27-G17. For every double-difference carrier phase observation and correctly determined ambiguities, the relative errors 2 mm see . The ambIn the second stage of the numerical tests, in the case of reinitialization at rvations , and, thThe properties of the functions iguities . TherefoThis work presents the new PREFMAR method for determining ambiguities in phase measurements for single measurement epochs, for GPS observations performed at L1 and L2 frequencies. Its efficiency mostly depends on the values of relative errors in DD phase observations. Ambiguity is selected based on a proprietary search function that uses the correlation between precisely determined ambiguity values depending on the frequency of satellite signals. The PREFMAR method allows determining ambiguities for individual measurement epochs without using a variance–covariance matrix with a float solution. For relative errors of 73 m see . Thus, tIn the case of relative errors of GPS float solution;Global code GPS/GNSS solution XYZ  and single-epoch double-differenced L1-L2 GPS measurements;A single-epoch of double-differenced L1-L2 GPS measurements.The developed PREFMAR method indicates ambiguity sets (Note that for case III, the PREFMAR method shows ambiguities even for a single DD observation, for any pair of satellites, but the accuracy of the code measurements is of key importance as we use double-difference observations from only two satellites. However, for much more accurate P5 code measurements transmitted at the L5 frequency, the III approach can be effectively used in L1-L5 positioning. The approach II seems to be the most universal, because in the global XYZ solution we can use any configuration of GNSS satellites with the most accurate code measurements and Kalman filter, which significantly increases the accuracy of the solution of the approximate XYZ position, both in static and kinematic positioning. Based on preliminary numerical results, the PREFMAR allows instantaneous ambiguity reinitialization if all satellites lost contact with a GNSS antenna.