Paraffin waxes are a promising material for heat storage with high energy density. Their low thermal conductivity, which limits the speed of charging and discharging in heat buffers, was previously shown to be improved by adding graphene nanofillers. In the present study, using molecular dynamics simulations, the segregation by molecular weight of polydisperse paraffin near graphene flakes is investigated. In liquid bidisperse paraffin composed of decane and triacontane, an aligned layer containing mainly triacontane was observed next to the graphene. Upon slow cooling, the wax crystallised into distinct layers parallel to the graphene sheet, with much stronger segregation by molecular weight than in the crystallised bidisperse wax without graphene. For polydisperse wax, the segregation effect was much less pronounced. The molten paraffin had a somewhat higher concentration of the longest chains in the first layers next to the graphene, but during crystallisation, the molecular weight segregation was only slightly increased. Measurements of crystallinity using an alternative version of the method developed by Yamamoto showed that the layers of wax were highly aligned parallel to the graphene, both in the solid state with all wax crystallised and in the liquid state with one layer of aligned wax above and below the graphene. Thermal conductivity was increased in planes parallel to the graphene flakes. The strong segregation of chain lengths in the bidisperse wax resulted in clear differences in thermal conductivity in the segregated regions. The less segregated polydisperse wax showed less variation in thermal conductivity. An important prerequisite for the ongoing energy transition is the storage of energy . This hoOne specific example of an organic PCM material is paraffin wax . BecauseMany previous studies, both experimental ,11 and mCompared to the TC of graphene (2000–4000 W/(m K) ), the obMany studies have been devoted to establishing a link between the crystallinity and thermal conductivity of polymers. For example, Borhani et al.  have stuZeng and Khodadadi  have carIn their theoretical studies, Wu et al.  and HariThe question of the molecular weight segregation of polydisperse polymer samples in the presence of nanofillers is still an open issue. It is well known that longer polymer chains have higher crystallisation temperatures ,23. UponComputational studies on the influence of paraffin–nanofiller interfaces were done by, e.g., Tong et al. . They peParaffin waxes used in practice usually are polydisperse. Knowing more about the interaction of polydisperse paraffin with graphene will provide insights into the behaviour of the composite material during melting–solidification cycles and enable further optimisation for use in heat storage. Using MD modelling, the interactions between paraffin wax and graphene fillers can be studied at the molecular scale, thereby giving information unobtainable by experiments. This is what we try to achieve in the present study.In the current paper, the influence of the graphene nanofiller on the segregation and resulting TC of the paraffin wax has been investigated. Our main goal is to have computational insights into the possible effects of the atomistically resolved nanofiller surfaces on the induced crystallisation, chain-length segregation and thermal conductivity for monodisperse and polydisperse paraffin-based nanocomposites. Segregation is quantified as the number of atoms belonging to molecules of each chain length per layer and the TC is measured by equilibrium MD (EMD), making use of the Green–Kubo formalism ,29, as wThe article is structured as follows. In In this section, we first consider the applied models for the paraffin–graphene composites and discuss measurement techniques afterwards.All simulations in the present study were performed with the fully atomistic Generalised Amber Force Field  (GAFF). us study  for the The paraffin molecules of different lengths needed to be produced separately, using AmberTools20 as described above. By combining the desired number of chains of each length, an initial configuration of the wax was produced that was very structured and had strong molecular weight segregation. To ensure a total loss of these, the paraffin was heated up to 600 K in the NVT ensemble, at which it was kept for 2 ns and then rapidly cooled down to 300 K. After adding the graphene sheet and the wax together, the composite was again heated to 600 K, held at this temperature for 2 ns and then rapidly cooled down to 450 K. This was done using the Berendsen thermostat and velocity rescale barostat at a pressure of 1 atm. Only the length of the box in the z-direction was allowed to change.Timesteps were set to 2 fs and C-H bonds were constrained using the LINCS  algorithGROMACS ,36 was uAs will be seen in Molecular weight segregation was quantified using relative densities. In each layer, the number density of atoms that were part of a chain of a specific length was determined. Dividing this by the average density (over the full simulation box) of the molecular weight under consideration, the relative density was obtained. This gives an indication of how segregated the chains of respective molecular weights are.Crystallinity was measured with the method developed by Yamamoto , altered(2σ)3  with σ tYamamoto . In addieSV [mT [K] the temperature. For ease of reading, only the definition of heat flux valid for two-body interactions is shown in Equation  measurement technique using the Green–Kubo ,29 formaJ→=1V∑ieiN of slabs. By interchanging the velocities of the coldest atom in the central slab—more specifically, slab number N/2 + 1 with N the total number of slabs—and the hottest atom in the first slab (at the edge of the simulation box), a temperature difference is induced. Once a steady state is reached, the heat flowing from the hot central slab to the cold outer slab is equal to the swapped kinetic energy,J [W/mvvm [kg] at each transfer, t [s] the total time of measurement and LLJ the heat flux [W/mAdditionally, the reverse non-equilibrium MD (RNEMD) technique developed by Müller-Plathe  was usedIn our previous publication , the TC Luo et al.  looked iThe effect of the presence of carbon nanofillers such as graphene on the thermal conductivity of monodisperse paraffin wax was studied, e.g., by Babaei et al. ,44. TheyIn Upon further cooling to 250 K, the decane has also crystallised into clear layers, as shown in The composite containing polydisperse wax at 450 K is shown in In In conclusion, a strong segregation was observed in the bidisperse composite. The presence of the graphene sheet induced the formation of much larger regions with a single chain length than was seen in the bidisperse wax without graphene. In the polydisperse composite, some segregation was present in the molten state. However, upon cooling, this did not increase as substantially as for the bidisperse wax. In comparison to the studies by Wu et al.  and HariApart from the segregation observed in bi-/polydisperse paraffin during cooling near a graphene sheet, the formation of crystalline structures is a striking aspect induced by the presence of the graphene sheet. This will be investigated next. Using a slightly altered version of the technique developed by Yamamoto , the cryIn In From previous studies ,45, it iSince, in the crystalline state, almost all molecules are perpendicular to the z-axis, it is expected that the z-component of TC will be reduced while heat transport along the xy-plane will be enhanced. Furthermore, it was shown previously ,45,46,47The most obvious method to measure TC appears to be the EMD technique using the Green–Kubo ,29 formaFurther TC measurements were done with the RNEMD technique developed by Müller-Plathe , as descIn the bidisperse composite, the same effects are observed. In the liquid state, the aligned layers close to the graphene have an increased TC compared to the fully liquid paraffin, which has a very low standard deviation in TC due to isotropy. In the semi-solid state, in which the triacontane has crystallised close to the graphene while the decane, which is concentrated further away, is still in a liquid state, there is almost a factor 2 difference in the TC of these regions. This agrees with expectation since the molecular weight and alignment are known to increase the TC ,22,45,46The polydisperse composite shows smaller differences in TC between the liquid and solid states. The molten paraffin at a large distance from the graphene shows isotropy, indicated by the very small standard deviation. The two layers closest to the graphene, the first of which has a significantly higher concentration of long molecules, as shown in In summary, in the liquid samples, alignment of the paraffin closest to the graphene sheet results in a higher TC compared to the remaining paraffin. The strong molecular weight segregation of the bidisperse wax during crystallisation results in a factor 2 difference in TC comparing the regions containing mostly long and mostly short molecules, respectively. The differences in the polydisperse paraffin were much less pronounced due to the less pronounced differences in composition.Paraffin waxes are a promising phase change material for heat storage, but significant improvement in their thermal conductivity is needed to enable fast charging and discharging. In the present study, we investigate the influence of a graphene nanofiller on the chain length segregation of polydisperse paraffin and the corresponding changes in TC. Making use of fully atomistic molecular dynamics simulations, the molecular weight segregation of paraffin as a consequence of the presence of graphene in the paraffin matrix, and the effect of this segregation on the thermal conductivity of the paraffin, were studied. With the paraffin in a molten state, a distinct layer above and below the graphene in which the paraffin molecules were highly aligned parallel to the graphene sheet was observed.Measurements of relative densities showed that this layer consisted mainly of the longer chains in the bidisperse paraffin. For the polydisperse paraffin, this effect was also observed, but found to be less pronounced, in agreement with the findings by Luo et al.  for paraA slightly modified version of the crystallinity measurement technique developed by Yamamoto , with whThermal conductivity was measured using two techniques. The equilibrium MD technique using Green–Kubo relations provided the qualitative confirmation that TC in the direction perpendicular to the graphene sheet was strongly reduced in the layered paraffin wax. Using the RNEMD technique developed by Müller-Plathe , TC was The layering in the wax implies an additional bottleneck for heat transport from graphene fillers into the paraffin and vice versa, in addition the limitation of interfacial thermal resistance between the two materials. The findings from this study provide a new direction for the optimisation of the TC of paraffin–graphene composites, suggesting to look into the functionalisation of the graphene surface.Additionally, measurement techniques for TC in the direction perpendicular to the planes of strongly layered materials could be further looked into.


Interactions between graphene, with its wide deployment in consumer products, and skin, the body's largest organ and first barrier, are highly relevant with respect to toxicology and dermal delivery. In this work, interaction of polyglycerol-functionalized graphene sheets, with 200 nm average lateral size and different surface charges, and human skin was studied and their potential as topical delivery systems were investigated. While neutral graphene sheets showed no significant skin interaction, their positively and negatively charged counterparts interacted with the skin, remaining in the stratum corneum. This efficient skin interaction bears a warning but also suggests a new topical drug delivery strategy based on the sheets' high loading capacity and photothermal property. Therefore, the immunosuppressive drug tacrolimus was loaded onto positively and negatively charged graphene sheets, and its release measured with and without laser irradiation using liquid chromatography tandem-mass spectrometry. Laser irradiation accelerated the release of tacrolimus, due to the photothermal property of graphene sheets. In addition, graphene sheets with positive and negative surface charges were loaded with Nile red, and their ability to deliver this cargo through the skin was investigated. Graphene sheets with positive surface charge were more efficient than the negatively charged ones in enhancing Nile red penetration into the skin. Positively and negatively charged graphene sheets interacted with human skin and enhanced penetration of Nile red-loaded into the epidermal and dermal layers of the skin. Moreover, graphene's cargo was released more efficiently into the skin layers after laser exposure. It has also been shown that passivation of graphene oxide by polyethylene glycol stimulates potent cytokine responses in peritoneal macrophages.27 In spite of such negative results and high toxicity, other reports showed no significant toxicity for polyethylene glycol-functionalized graphene sheets, with efficient renal and fecal excretion. Such contradictory results may be due to differences in physiochemical properties of the studied graphene sheets, including size, functionality and surface charge.28 Skin, as the largest and first body's organ is at high risk upon exposing to graphene derivatives. However, our knowledge regarding interactions between skin and graphene derivatives mostly is limited to in vitro studies including interactions with skin fibroblasts,29 keratinocytes and human epidermis.30–32 It has been shown that exposing of HaCaT skin keratinocytes to few layers of graphene derivatives for a short time results in reversible toxicity, i.e., these cells were able to recover from the toxic effects. Moreover, it has been demonstrated that graphene derivatives exfoliated by surfactants induce skin irritation, while others do not provoke an adverse effect upon single exposure.33,34 Considering graphene materials for dermal application, it is essential to address their skin sensitization and irritation as a possible adverse outcome. Recently, an in vivo study suggested dermal exposure of graphene oxide (GO) and few-layer graphene (FLG) has no irritation or sensitization effect on skin immune cells.35As the waste products of graphene derivatives are discharged in greater amounts, due to their production in larger scales for a growing range of applications, the health risks of occupational and environmental exposure to these materials grow in importance.36 They have been used for loading therapeutic agents and targeting these cargos to tumor sites.37 Hyperbranched polyglycerol and polyglycerol-based nanomaterials are hydrophilic polyfunctional vectors with low immunogenicity and high potential for future biomedical applications.38,39 Passivation of graphene sheets with polyglycerol has resulted in two-dimensional platforms with unique physicochemical properties and high potential for future biomedical applications.40–44 Interactions of polyglycerol-functionalized graphene sheets with cells, their cellular uptake pathways, and even their journey inside the cells depend strongly on their functionality, size and surface charge.45,46 Considering these factors, polyglycerol-functionalized graphene sheets have been used for various biomedical applications, including pathogen incapacitation and tumor therapy.43,47 Moreover, polyglycerol and similar polymeric nanocarriers have been used as topical drug delivery systems abundantly,39,48,49 but their main disadvantage is the lack of a stimuli-responsive factor to trigger the release their cargo in the target tissue. Hybridization of polyglycerol with graphene results in new systems, which can respond to laser as a triggering factor. In addition, graphene will improve interactions of polyglycerol derivatives with the biosystems, as it has been observed for single cells.45,50On a positive side and in the less-toxic concentration range, polymer-functionalized graphene sheets are interesting candidates for biomedical applications due to their high loading capacity, fast cellular uptake, and photothermal property.Accordingly, polyglycerol-functionalized graphene sheets can be used as new topical drug delivery systems, owing to their high loading capacity for hydrophobic drugs such as tacrolimus (TAC). Acceleration of the rate of release of their cargo in the target tissue decreases their side effects and increase their therapeutic efficiency. Moreover, they can induce heat in the target layer of skin, resulting in photothermal therapy. Due to such properties, polyglycerol-functionalized graphene sheets can be used for the sustainable delivery of topical drugs. They can enhance the bioavailability, stability and skin penetration of drugs, which in turn increase their therapeutic effects.This work uses microscopic techniques to investigate interactions between human skin and polyglycerol-functionalized graphene sheets with 200 nm lateral size and different surface charges . While n51 Hyperbranched polyglycerol (hPG), with Mn ≈ 10 000 g mol−1, was synthesized via one-pot, ring-opening anionic polymerization (ROAP).52 hPG covered TRGO (hPG-Gs), hPG-amine covered TRGO (hPGN-Gs) and hPG-sulfate covered TRGO (hPGS-Gs) with a size of around 200 nm were prepared according to our previously published articles.45,46 IDCC NHS-ester  was bought from IC Discovery GmbH. Cell counting kit-8 (CCK-8) assay, Hoechst 33342 were purchased from Thermo Fisher Scientific. Milli-Q water was used in all experiments. Phosphate-buffered saline (PBS) (10×) pH 7.4 (Thermo Fisher Scientific) was diluted 10 times with Millipore quality water.Thermally reduced graphene oxide (TRGO) was synthesized according to the method in literature.53 by means of an Agilent 1260 Infinity LC system coupled to an Agilent 6490 triple quadrupole-mass spectrometer  interfaced with an electrospray ion source operating in the positive ion mode (ESI+).Atomic Force Microscopy (AFM) was measured with a MultiMode Nanoscope V scanning probe microscopy (SPM) system  in air under ambient conditions. Zeta potential data were obtained in PBS solution by Malvern (UK) NANO ZSPO. For transmission electron microscopy (TEM) a 5 μL droplet of sample solution was placed on a hydrophilized carbon film covered copper grid (400 mesh). The supernatant fluid was removed by blotting with a filter paper and the sample was allowed to dry in air. A standard holder was used to transfer the dried samples into a Talos L120C transmission electron microscope  operated at an acceleration voltage of 120 kV. Micrographs were recorded with a 4k × 4k Ceta 16M camera. The near-infrared (NIR) laser irradiation was performed with a diode infrared laser module (808 nm), Changchun New Industries (China). CCK8 assay was measured with a TECAN (Switzerland) Infinite M200 Pro microplate reader. Confocal laser scanning microscopy (CLSM) was conducted by Leica microscope (Germany) TCS SP8 (63× oil-immersion objective lens). Tacrolimus quantification by stable-isotope dilution liquid chromatography tandem-mass spectrometry (LC-MS/MS) was performed as reported2O): λmax: 500 nm (1 mg mL−1).hPG-Gs  was dissolved in dry DMF (10 mL). IDCC  was added, and the solution was stirred for 48 h at 60 °C. The solution was cooled to room temperature and dialyzed against deionized water for 48 h to obtain the IDCC – . IDCC labeling of hPGN-Gs and hPGS-Gs were performed with the same procedure UV/vis (H1 mg hPG-Gs was dispersed in 10 mL PBS. Then 2 mg TAC (dissolved in 0.5 mL ethanol) was added dropwise into the GH dispersion under stirring at room temperature. The mixture was stirred for 1 h before it was centrifuged at 2000 rpm for 5 min. The supernatant was dialysis in Milli-Q for 24 h to get TAC-loaded hPG-Gs and the precipitate was collected for the measurement of loading capacity. The precipitate was dissolved in 10 mL ethanol. Then the ethanol solution was centrifuged at 5000 rpm for 10 min and 1 mL supernatant was extracted. Subsequently, the solvent was evaporated and the dried TAC was dissolved in acetonitrile for HPLC measurement to determinate the TAC concentration. TAC-loaded hPGN-Gs and TAC-loaded hPGS-Gs could be obtained with the same procedure. The loading capacity of hPG-Gs, hPGN-Gs and hPGS-Gs was determined by the HPLC results.−1) of Nile red in methanol was added to a PBS solution of hPG-Gs, hPGN-Gs and hPGS-Gs dropwise, while mixture was gently stirred . It was stirred for additional 2 h at 25 °C and then methanol was evaporated. Subsequently, hPG-Gs, hPGN-Gs and hPGS-Gs loaded with Nile red were dried at 50 °C for 1 h. The obtained compound was dissolved in MilliQ water and stirred for additional 10 min at room temperature (25 °C). The mixture was centrifuged for 10 min at 3000g and the supernatant was collected and lyophilized. UV/vis (methanol): λmax: 350 nm (0.001 mg mL−1).A 60 μL stock solution  stirred at 500 rpm. After 30 min, 36 μL of the test formulations and cream was administrated onto the skin surface (finite-dose approach) and incubated for 18 h. Furthermore, after 6 h of incubation time hPGN-Gs and hPGS-Gs samples were irradiated with NIR laser  continuously over 30 min for a total exposure time of 2.5 min and samples were remained for another 12 h. Subsequently, the treated skin areas were punched, embedded in tissue freezing medium and stored at a low temperature (−80 °C). To determine Nile red penetration, the skin was cut vertically into 10 μm thick slices using a cryotome. Subsequently, the slices were subjected to normal and fluorescence light .To investigate the interaction of graphene derivatives and topical drug delivery efficiency, IDCC-labelled or nile red-loaded (0.001% w/w) hPG-Gs, hPGN-Gs and hPGS-Gs was studied on excised human skin  and in accordance with the Declaration of Helsinki guidelines. The 55 skin samples were fixed in Karnovsky solution  overnight, washed in 0.1 M cacodylate buffer , and incubated in 1% osmium tetroxide  for 4 h. Samples were dehydrated in an ascending series of ethanol followed by the intermedium propylene oxide . The specimens were subsequently embedded in a mixture of Agar 100 (epoxy resin), DDSA (softener), MNA (hardener) and DMP 30  . Polymerization was done at 45 °C and 55 °C, each for 24 hours. Semi- and ultrathin sections were cut using an Ultramicrotome Reichert Ultracut S . Semi-thin sections (0.5 μm) were stained with modified Richardson solution for 45 seconds on an electric hot plate adjusted to 80 °C. Semithin sections were examined with a light microscope Olympus CX21 . Next ultrathin sections (80 nm) were mounted on nickel grids , contrasted with Uranyless EM stain followed by Reynolds lead citrate 3%  and examined with a transmission electron microscope JEM-1400 plus .After application of the graphene sheets according to our previously reported paper,Ultrathin sections were mounted on Formvar filmed grids and frozen before the transfer into the transmission electron microscope by the use of a Vitrobot™ Mark IV . The Vitrobot was operated at 22 °C and at a relative humidity of 100%. The grids were plunged without further treatment into liquid ethane just above its freezing point and transferred in liquid nitrogen. The grids were “clipped” (= inserted in a rigid clip-ring) and transferred under liquid nitrogen into a Talos Arctica™ TEM (Thermo Fisher Scientific) operated at an accelerating voltage of 200 kV and at a nominal magnification of 4300× or 2600×, respectively. Image series in the tilt range of −64°/64° (2° tilt-increments) were recorded at full 4k resolution resulting in a pixel size of 4.91 nm or 8.01 nm, respectively. The defocus value was set to −20 μm. Image stack alignment and 3D reconstruction were performed in the context of the Inspect 3D software V4.3 (Thermo Fisher Scientific). Stacks of 10 image slices from the reconstructed volume were exported for data presentation.2 at 37 °C. CCK8 assay. HaCat cells (1 × 104 cells per well) were seeded in 96-well plates with 100 μL DMEM and incubated for 24 h before the tests. hPG-Gs, hPGN-Gs and hPGS-Gs in the culture medium were added to the medium-removed 96-well plates with different concentrations and incubated for another 24 h, respectively. After that, the culture medium solutions were removed and the cells were rinsed with PBS twice. 100 μL culture medium with 10 μL CCK8 solution was added to each well. After incubation for the following 2 hours, 75 μL medium was carefully transferred to a new plate to avoid the influence of graphene on final results, and the absorption was measured at wavelength of 450 nm with a microplate reader. Cells without any treatments were regarded as a negative control. Each sample was measured 3 times.All cell experiments were conducted according to German genetic engineering laws and German biosafety guidelines in the laboratory (level 1). Dulbecco Modified Eagle Medium (DMEM) (Gibco) and fetal bovine serum (FBS) were applied for the following experiments. HaCat cells were cultured in DMEM supplemented with 10% (v/v) FBS and 1% penicillin. The cells were incubated in a humidified atmosphere with 5% CO−1), washed twice with PBS, and covered with fresh cell culture medium (DMEM). Confocal images were taken with an inverted confocal laser scanning microscope Leica DMI6000CSB SP8  with a 63×/1.4 HC PL APO CS2 oil immersion objective using the manufacturer-given LAS X software in sequential mode with the following channel settings: transmission Ch , excitation laser line 405 nm, detection of transmitted light (photomultiplier); Ch1 (Hoechst 33342): excitation laser line 405 nm, detection range 410–484 nm (hybrid detector); Ch2: excitation laser line 488 nm, detection range 493–712 nm (hybrid detector).The uptake of IDCC-labeled hPGN-Gs and hPGS-Gs by HaCat cells was analyzed using confocal laser scanning microscopy (CLSM). The cells were propagated as described above. For CLSM, HaCat cells were seeded in eight-well ibidi slides (ibidi treat) in 270 μL of DMEM. After cell attachment for 4 h to 24 h, 30 μL of a postseeding solution containing compound was added, and the cells were further incubated overnight. Before imaging, the cells were stained with Hoechst 33342 (1 μg mL−2) for 5 min every 2 h. The amount of released TAC at different time frames was determined by LC-MS/MS method. To this end, lyophilized samples were resuspended in acetonitrile containing 1 μM stable-isotopically labeled TAC. Samples were then shaken at 200 rpm for 1 h before particles were allowed to sediment for 1 h and supernatants were analyzed. The drug release studies were performed in triplicates.hPGN-Gs and hPGS-Gs with 50 μg TAC content was dispersed separately in PBS , and equally transferred into 2 dialysis tubes (spectra/Por MWCO 2 kD). Then, the tubes were immersed in 2 vials containing 30 mL PBS (pH 7.4). Vials were set up in a thermostatic water bath (37 °C) on a heating rotator. 5 mL solution from PBS medium out of dialysis bag were collected at each 2 h intervals, and meanwhile, fresh PBS was replenished. One of the vials was irradiated with NIR laser  and then equally distributed into 2 tubes. Subsequently, near-infrared (NIR) laser irradiation  was performed to these tubes for 5 min. The temperature of the solutions in tubes were recorded by IR thermal imaging camera .hPGS-Gs and hPGN-Gs were dispersed in 2 mL PBS  at 100 μg mL−1. Afterwards, the uptake of the functionalized graphene sheets, hPGN-Gs and hPGS-Gs, by HaCat cells at different time frames was monitored. According to our previous study, hPG-Gs showed no significant uptake due to the polyglycerol coverage.40–43 While the negatively charged counterparts with sulfate functional groups showed considerable uptake after 24 h  for 3 minutes increased their temperature to 65 °C , a topical therapeutic agent for treating T cell-mediated diseases such as eczema and psoriasis. TEM images of hPGN-Gs and hPGS-Gs after loading with tacrolimus showed sheets with comparable dimensions as before drug loading  for tacrolimus, and they were able to release the drug under short-term NIR laser irradiation. In addition, skin penetration experiments of Nile red-loaded hPGN-Gs and hPGS-Gs confirmed that NIR irradiation increases the release of Nile red inside the skin. In particular, hPGN-Gs enhanced the Nile red's penetration in the viable skin layers more efficiently than hPGS-Gs. Therefore, the potential health risk of graphene should be considered and investigated further, e.g. in in vivo experiments. Outcomes of this work shed a light in the future biomedical applications and toxicity of similar carbon based nanomaterials including fullerene, carbon nanotubes and graphitic materials upon exposure to human skin.This study evaluates possible interactions between of functionalized graphene sheets with lateral size 200 nm and different surface charge – hPGN-Gs (+30 mV), hPG-Gs (0 mV) and hPGS-Gs (−30 mV) – and human skin. As a first step, we evaluated cytotoxicity and cellular uptake of these materials against HaCat cells for 24 h incubation time. The results demonstrate that hPGN-Gs, hPG-Gs and hPGS-Gs do not show significant toxicity up to 30 μg mLex vivo skin studies, data interpretation, writing—original draft preparation, visualization, F. Z.; synthesis, in vitro cell studies, drug loading and release studies Z. T.; LC-MS/MS measurements, data interpretation, visualization, methodology, F. S.; TEM imaging from graphene sheets, data interpretation, visualization, K. L.; TEM imaging from skin samples, data interpretation, visualization, S. K. and C. B.; ex vivo studies, data interpretation, visualization, F. R.; conceptualization, guidance, resources, funding acquisition, B. K.; J. P.; S. H.; A. V. and R. H. All authors have read and agreed to the published version of the manuscript.Conceptualization, methodology, There are no conflicts to declare.NA-005-D3NA00574G-s001


ChatGPT (OpenAI) has gained considerable attention because of its natural and intuitive responses. ChatGPT sometimes writes plausible-sounding but incorrect or nonsensical answers, as stated by OpenAI as a limitation. However, considering that ChatGPT is an interactive AI that has been trained to reduce the output of unethical sentences, the reliability of the training data is high and the usefulness of the output content is promising. Fortunately, in March 2023, a new version of ChatGPT, GPT-4, was released, which, according to internal evaluations, was expected to increase the likelihood of producing factual responses by 40% compared with its predecessor, GPT-3.5. The usefulness of this version of ChatGPT in English is widely appreciated. It is also increasingly being evaluated as a system for obtaining medical information in languages other than English. Although it does not reach a passing score on the national medical examination in Chinese, its accuracy is expected to gradually improve. Evaluation of ChatGPT with Japanese input is limited, although there have been reports on the accuracy of ChatGPT’s answers to clinical questions regarding the Japanese Society of Hypertension guidelines and on the performance of the National Nursing Examination.The objective of this study is to evaluate whether ChatGPT can provide accurate diagnoses and medical knowledge for Japanese input.Questions from the National Medical Licensing Examination (NMLE) in Japan, administered by the Japanese Ministry of Health, Labour and Welfare in 2022, were used. All 400 questions were included. Exclusion criteria were figures and tables that ChatGPT could not recognize; only text questions were extracted. We instructed GPT-3.5 and GPT-4 to input the Japanese questions as they were and to output the correct answers for each question. The output of ChatGPT was verified by 2 general practice physicians. In case of discrepancies, they were checked by another physician to make a final decision. The overall performance was evaluated by calculating the percentage of correct answers output by GPT-3.5 and GPT-4.Of the 400 questions, 292 were analyzed. Questions containing charts, which are not supported by ChatGPT, were excluded. The correct response rate for GPT-4 was 81.5% (237/292), which was significantly higher than the rate for GPT-3.5, 42.8% (125/292). Moreover, GPT-4 surpassed the passing standard (>72%) for the NMLE, indicating its potential as a diagnostic and therapeutic decision aid for physicians.GPT-4 reached the passing standard for the NMLE in Japan, entered in Japanese, although it is limited to written questions. As the accelerated progress in the past few months has shown, the performance of the AI will improve as the large language model continues to learn more, and it may well become a decision support system for medical professionals by providing more accurate information. ChatGPT based on GPT-3.5 was launched by OpenAI in 2022 and has become sensational owing to its capacity to respond to any question using natural language. GPT-3.5 has 175 billion parameters in its language model, while GPT-4, introduced a few months later, did not disclose its number of parameters . The useThe usefulness of ChatGPT in English is widely appreciated . It is aThe objective of this study is to evaluate whether ChatGPT can provide accurate diagnoses and medical knowledge from Japanese input. We input questions from the NMLE into GPT-3.5 and GPT-4 in Japanese and validated the responses. We assumed that the system would output medical information with a high degree of accuracy, even if the input is in Japanese, as its usefulness is increasingly being acknowledged in many fields.Questions from the National Medical Licensing Examination (NMLE) in Japan, administered by the Japanese Ministry of Health, Labour and Welfare (MHLW) in 2022, were used. All 400 questions were included. Figures and tables that ChatGPT could not recognize were excluded, and 292 questions with only text questions were selected. We instructed GPT-3.5 and GPT-4 to input the Japanese questions as they were and to output the correct answers for each question. The output of ChatGPT was verified by 2 general practice physicians. When there were discrepancies, they were checked by another physician to make a final decision. The overall performance was evaluated by calculating the percentage of correct answers output by GPT-3.5 and GPT-4.Because the data studied by GPT-3.5 and GPT-4 were text data available on the internet until September 2021, the target for evaluation was the NMLE held in February 2022, for which the MHLW officially published the questions and answers . There aAs ChatGPT does not allow for the input of images and tables, these questions were excluded (102 questions). In addition, questions that are officially designated as “unscored” by the MHLW, which sponsors the NMLE, were excluded (6 questions). Unscored questions were excessively difficult in terms of the test difficulty level or had errors in their formulation, resulting in situations where the selection of a single answer is impossible for single-choice questions, among other cases. Finally, 292 questions that could be used to accurately assess ChatGPT were included .GPT-3.5 was used on January 30, 2023, and GPT-4 was used on March 24, 2023. ChatGPT is an AI language model based on the GPT architecture, which is a type of neural network designed for natural language processing tasks. It generates responses on the fly based on the likelihood of the next word, given the relationships between words within the neural network learned during training. ChatGPT is specifically designed for conversational interactions. It can engage with users, answer questions, and sustain conversations. By analyzing the preceding text and context, ChatGPT generates responses aimed at mimicking human-like conversations.In the context of ChatGPT, prompts containing target questions are inputted. A new chat was created for each question to prevent the effect of prior input questions . Each quThis study did not involve human or animal participants and ethics approval was not required.In GPT-4, 227 of the 292 questions were answered correctly in 1 attempt (77.7%), 47 were answered incorrectly (16.1%), and 18 (6.2%) of the questions were not completed in the form of answers . Specifically, although 1 answer had to be chosen from a list of options, 2 answers were selected for 8 questions, 3 for 4 questions, 4 for 3 questions, and no answers for 3 questions. Finally, answers to these questions were obtained by adding a “choose one” option to the question. The overall correct response rate was 81.5% (237/292); 88.1% (74/84) for the required questions, 75.4% (89/118) for the comprehensive questions, and 82.2% (74/90) for the specific questions.In GPT-3.5, 102 questions were correctly answered in 1 attempt (34.9%), 143 questions incorrectly (50%), and 47 (16.1%) were not completed in the form of answers . Specifically, regarding the questions where 1 answer was chosen from a list of options, 2 answers were given for 3 questions, 3 for 7 questions, 5 for 3 questions, and no answers for 34 questions. For these questions, we added the option of choosing 1 answer. For the 22 questions that did not receive an answer because there were multiple queries in response to the question text, the queries were inputted separately into the chat box and answers were obtained. The correct response rate was 42.8% (125/292) overall, 50% (44/84) for the required questions, 40.7% (48/118) for the comprehensive questions, and 38.9% (35/90) for the specific questions .In addition to answering the choices, the output included secondary information related to the medical vocabulary in the explanations and question text. These were found in 94 questions in GPT-3.5 and 255 questions in GPT-4. As shown in The primary outcome of this study was the ability to accurately understand medical information by inputting Japanese prompts, and the NMLE was tested as a method of evaluating this ability. This study evaluated whether the selected option was the correct answer. The number of correct answers was calculated and evaluated, first to see if ChatGPT could obtain a score percentage that would pass the examination. GPT-4 was above the passing standard for the NMLE  while GPT-3.5 was below. These results are similar to those of a previous study that tested the performance of ChatGPT on the USMLE . We did We have not been able to evaluate the image and table questions because they are not supported in GPT-3.5 and cannot be inputted in GPT-4 at this time. If all ChatGPT versions could answer the 102 questions containing figures and tables, and the answers were incorrect, it is possible that the passing criteria would not be met. However, many questions that include images and tables use them in addition to clinical information, and it remains possible that the correct answer could have been given based on the question text, even if the images and tables were not discussed. The versions used in this study, namely, GPT-3.5 and GPT-4, were evaluated as of January 30, 2023, and March 24, 2023, respectively. Further updates are expected in the future, and they should be continuously evaluated.GPT-4 reached the passing standard for the NMLE in Japan with prompts entered in Japanese, although it is limited to text-only questions. As the accelerated progress in the past few months has shown, the performance of the AI will improve as the large language model continues to learn more, and it may well become a decision support system for medical professionals by providing more accurate information.


The polyimide polymer was the source material for the fabrication of the LIG. The doping process was performed in situ using the CO2 laser, which works as a rapid thermal treatment to combine graphene and NiO particles. NiO was used to improve the capacitance of graphene by combining an electric double-layer capacitor (EDLC) with the pseudo-capacitance effect. The high-resolution transmission electron microscopy, energy-dispersive X-ray spectroscopy, and Raman spectroscopy showed that the structure of the LIG is multilayered and waved. The HRTEM image proves the distribution of NiO fine particles with sizes of 5–10 nm into the graphene layers. The electrochemical performance of the as-prepared LIG was tested. The effect of the combination of the two materials (oxide and carbon) was investigated at different concentrations. The LIG showed a specific capacitance of 69 Fg−1, which increased up to 174 Fg−1 for the NiO-doped LIG. The stability investigations showed that the electrodes were very stable for more than 1000 cycles. This current study establishes an innovative method to improve the electrochemical properties of LIG.Herein, we have reported a novel strategy for improving the electrochemical performance of laser-induced graphene (LIG) supercapacitors (SCs). The LIG was prepared using a CO It has been suggested that SCs are an alternative solution for single and hybrid applications compared with other storage devices . In addiSCs are categorized into electric double layer capacitors (EDLC)  and pseu2 hybrid orbitals in a hexagonal honeycomb lattice. The combination of these unique physicochemical properties makes graphene-based materials promising candidates for electrochemical energy storage devices [Graphene material has interesting properties due to its high electrical conductivity, high surface area, mechanical strength, and lightweight ,8. There devices ,12,13,142 lasers has been reported [2/g [A method to produce multilayer graphene (laser-induced graphene (LIG)) from polyimide polymer films using COreported ,16. Laseted [2/g ,16. In a2 laser technology. The structure of the LIG was studied using electron microscopy, EDX, and Raman spectrometry. LIG and doped LIG were investigated as supercapacitor electrodes. The performance of the LIG electrodes was significantly improved by NiO doping. An extensive study was carried to analyze the electrochemical properties of the manufactured electrodes. However, one must keep in mind that increasing the capacitance value does not mean only a modification of the intrinsic capacitance of the electric double layer. The higher capacitance of the capacitors may result from combining the pseudo-capacitance and EDLC mechanisms of charge storage. This type of capacitor is often confused with typical redox-based storage. In this work, we present an easy and simple method to improve the storage properties of graphene by merging NiO directly using CO2, corresponding to 2.5 mL of NiO-suspended solution, and 2.0 mg/cm2, corresponding to 5.0 mL of NiO-suspended solution, were wisely dropped on 5.0 cm2 of the fabricated LIG sheet during drying at 60 °C on a hot plate. Afterward, the sheet was again subjected to the laser beam under the same conditions. The steps of the LIG preparation are shown in 2 and 2.0 mg/cm2, three electrodes termed LIG-0, LIG-1, and LIG-2 were prepared, where LIG-0 stands for pristine LIG, LIG-1 stands for NiO-doped LIG with 1.0 mg/cm2 of NiO, and LIG-2 stands for NiO-doped LIG with 2.0 mg/cm2 of NiO.The suspended NiO was prepared based on the prescribed NiO amount using ethanol. NiO nanopowder with a particle size of less than 50 nm was purchased from Sigma Aldrich and was used without further modification. An amount of 10 mg NiO was added to 5 mL of ethanol at a concentration of 2 mg/mL. The suspended solution of NiO was sonicated for 20 min before withdrawing the required amount. The laser system contained 40 W of the maximum power with a head speed of 400 mm/s. Polyimide sheets  with a thickness of 5.0 mil (170 µm and 10 mm × 50 mm) were used as raw materials for LIG. To fabricate the LIG, the laser power was 9.0 W and the head speed was 120 mm/s. Then 1.0 mg/cm−1 were used for measuring the cyclic voltammetry curves. The electrodes were charged and discharged (GCD) at current densities of 1.6 to 3.3 Ag−1.A high-resolution transmission electron microscope  working at 200 kV was used to analyze the products. For TEM measurements, the sample was dropped on a carbon-coated copper grid after the sonication in ethanol. Selected area electron diffraction (SAED) was carried out for the pristine and doped graphene. The Raman shift of the graphene was carried out using LabRAM-HR800 of a He-Cd laser with a wavelength of 633 nm. The supercapacitor measurements were measured using the CorrTest electrochemical workstation. The experiments were carried out in a three-electrode electrochemical cell including a counter electrode of Pt wire, a reference electrode of saturated calomel electrode (Ag/AgCl), and a working active electrode. The aqueous electrolyte was 1.0 M KOH. Within a frequency range of 0.1 Hz to 1.0 MHz, the electrochemical impedance spectrum (EIS) was examined. Various swipe voltages of 5 to 100 mVs2 laser. The lattice image of the LIG with a wide scale shown in For more deep information about the NiO particle size and distribution on the graphene surface, HRTEM images were examined for the NiO-doped graphene sample, as shown in 2g of the sp2 bond in the Brillouin zone center [−1, which could be assigned to disordered graphitic carbon and the presence of sp3 defects [−1 for the LIG. The Raman spectra of the NiO-doped LIG are also shown in −1 [−1 and longitudinal optical (LO) at 530 cm−1) and two phonons  excitations. For pristine LIG, the intensity ratios of the D/G and 2D/G bands are important parameters, which give more details about the nature of LIG, where ID/G indicates the level of the defect in LIG, and I2D/G indicates the number of layers. The ID/G value was 0.29, which confirms the low lattice defects of the LIG prepared here [2D/G showed a value of 0.43, which is less than 1.0 [The multilayer LIG was well defined by the Raman spectrum, where only three D-, G-, and 2D-bands were observed. These three bands are shown in the Raman spectrum im e center . The dis defects . The secwn in −1 ,22, whilwn in −1 . Raman sred here ,24,25. Tthan 1.0 .−1, as shown in 2 of NiO, redox peaks appeared at a value of 0.52 and 0.2 V. There was also a broadening in the region between 0.4 and −0.7 V, as shown in Electrochemical measurements were carried out in a three-electrode configuration in an alkaline KOH electrolyte. The application was carried out for the three prepared electrodes  under the same conditions to evaluate their performance. Cyclic voltammetry (CV) curves were investigated at various scan rates between 5 and 100 mVs−1, where the higher current density exhibited faster charging, and vice versa. The maximum charging–discharging time of LIG-0 at 1.6 Ag−1 was 70 s, while it was 107 and 112 s for LIG-1 and LIG-2, respectively. However, it was observed that the LIG-2 electrode took a longer time (67 s) to be charged compared to LIG-1 (57 s), which took a longer time compared to the LIG-0 electrode (41 s). The observed behavior was due to the chemical modification of the LIG by NiO, where physical adsorption between the LIG material and NiO nanoparticles occurred. Thus, the electrode has a performance of oxidation and carbon materials, where the oxide and carbon materials perform both the pseudo-capacitance and EDLC mechanisms. The scribing of NiO by the laser generated a modification for the surface of the LIG. Thus, an increase in the charge capacity of the modified LIG by 200–300% compared to that of the raw LIG was observed.The behavior observed in the above CV curves was reflected and shown in the galvanostatic charge–discharge (GCD) curves, as shown in s) containing the resistance of the electrode materials, the ionic resistance of the electrolyte, and the contact resistance between the electrode and the current collector; and (2) the radius of the semicircle represents the electrode conductivity and the charge transfer resistance (Rct) of the electrode material. In the middle-frequency region, the oblique curve represents the Warburg impedance, which represents the diffusion/transport of ions in the electrolyte. The capacitive behavior of the electrode takes place on the curve in the low-frequency region. As we mention later, the electrochemical properties of graphene changed significantly due to NiO doping, as the capacitance increased to 174 F/g. The modeled values of Rs and Rct withdrawn from the Nyquist diagram are listed in t also showed a large change after doping, where it increased from 5 up to 22 Ω. The interesting is the semi-circle, which was largely demonstrated at a higher frequency for the doped electrodes. It was expected that in the case of pure LIG, the electrode would work as an EDLC supercapacitor, as indicated in the CV and GCD curves. In the EDLC mechanism, the electrolyte ions accumulate on the electrode surface to form a double layer charge. However, in the LIG doped with NiO, both pseudo-capacitance and EDLC mechanisms occurred.The electrical impedance spectroscopy (EIS) was measured in the range of 0.1 Hz to 1.0 MHz. The three electrodes presented typical AC impedance attributes of supercapacitors. −1 when ionic liquid, organic, and aqueous electrolytes were used [−1 at 5 mVs−1 for LIG-0 and decreased with an increase in the scan rate. The specific capacitances of the LIG-1 and LIG-2 were 152 Fg−1 and 174 Fg−1, respectively. It was observed that the NiO-doped LIG was still higher than that of the pristine LIG at all scan rates. −1 are 150, 148, and 52 Fg−1 for LIG-2, LIG-1, and LIG-0, respectively. With increasing the discharging current up to 3.3 Ag−1, the specific capacitances were 58, 32, and 15 Fg−1 for LIG-2, LIG-1, and LIG-0, respectively.The specific capacitance of graphene has been previously reported to be 75, 99, and 135 Fgere used ,28. Figu−1 and a specific power of 429 Wkg−1, the specific energy has maximum values of 5.3, 5.6, and 2.0 Whkg−1 for LIG-2, LIG-1, and LIG-0, respectively. The specific energy of the NiO-doped LIG increased about three times compared to that of LIG-0. With increasing current density, the specific energy decreased, while the specific power increased.Supercapacitors, electrochemical capacitors, double layer capacitors, or Farad capacitors are electrochemical components for storing energy through a symmetric electrode. It is a kind between a conventional capacitor and a battery, adopting a double layer and a pseudo-capacitor to store electrical energy. It has a high electrical power compared to batteries, which have a high energy. Often, in capacitors, there is no chemical reaction during the energy storage process, and thus it is reversible and enables us to recharge and discharge thousands of times. Supercapacitors have fast charging speeds and can reach more than 95% of their capacity within a short time. −1 is shown in −1 for the rest of the cycles. As shown in −1. It is obvious that LIG-0 started with high efficiency at 95% and continued at almost the same CE until the 1000th cycle. For LIG-1, the CE started at 90% for LIG-1 and improved with the 300th cycle to 96% to the 1000th cycle. For LIG-2, the CE value was 77% and gradually improved to 92% at the 1000th cycle. Despite doping graphene with a material that exhibits a Faradaic reaction, the electrodes still show a good electrochemical performance in terms of cycling, charging, and discharging.The specific capacitance measured for 1000 cycles of charging and discharging at 2.3 Ag2 laser can be proposed. As shown in the CVs of LIG-0, LIG-1, and LIG-2 presented in 2+ concentration due to the increase in NiO/graphene, which exposes the pseudocapacitive behavior. However, it is expected that there will be a trade-off between the conductivity  and pseudocapacitance (based on NiO) in the presence of extra NiO, which may lead to an increase in the resistance and reduce the current. It is observed in The doping mechanism of NiO-doped graphene through the CONiOOH+e− ,32. Thus2 at different charging currents or scan rates of the swipe voltage. Also, the high specific capacitance of LIG-PEDOT recorded a value of 115 F/g, compared to the present value of 174 F/g for NiO-doped LIG.2 laser. HRTEM confirmed the fabrication of multilayered graphene sheets using a CO2 laser. It has been clearly shown that the crystalline planes appear (002) mostly in this type of graphene, with a d-space of 0.33 nm. Also, through SAED, it was found that this graphene contained two planes of (002) and (100). The fabrication of multilayer graphene and NiO-doped graphene sheets was also confirmed by Raman spectra, where D, G, and 2D of graphene and scattering peaks of NiO. Electrodes with different concentrations of NiO were tested for their electrochemical properties as supercapacitors without any additional processing. A significant improvement was observed in the performance of the composite electrodes as the specific capacitance increased from 69 to 174 Fg−1 at a scanning rate of 5 mVs−1. The same performance was also observed in the case of the specific capacitance calculated from the charge and discharge curves, which had that the two concentrations of NiO showed almost the same specific capacitance values. In all electrodes, the lifecycle performance was excellent up to the thousandth cycle. The stored energy increased 3 times (from 2.0 to 5.6 Whkg−1 corresponding to a power of 429 WKg−1). We can conclude that the current combination strategy between oxide and graphene is suitable for improving the electrochemical properties of graphene and can be used for further studies in the future.In summary, we summarize an easy strategy to improve the capacitive performance of graphene by incorporating nickel oxide into its layers using direct laser technology. NiO fine particles of less than 50 nm were dropped on the fabricated graphene and scribed using a CO


Assessing the fidelity of intervention components enables researchers to make informed judgements about the influence of those components on the observed outcome. The ‘Implementing work-related Mental health guidelines in general PRacticE’ (IMPRovE) trial is a hybrid III trial aiming to increase adherence to the ‘Clinical Guidelines for the diagnosis and management of work-related mental health conditions in general practice’. IMPRovE is a multifaceted intervention, with one of the central components being academic detailing (AD). This study describes the fidelity to the protocol for the AD component of the IMPRovE intervention.All AD sessions for the trial were audio-recorded and a sample of 22% were randomly selected for fidelity assessment. Fidelity was assessed using a tailored proforma based on the Modified Conceptual Framework for fidelity assessment, measuring duration, coverage, frequency and content. A descriptive analysis was used to quantify fidelity to the protocol and a content analysis was used to elucidate qualitative aspects of fidelity.A total of eight AD sessions were included in the fidelity assessment. The average fidelity score was 89.2%, ranging from 80 to 100% across the eight sessions. The sessions were on average 47 min long and addressed all of the ten chapters in the guideline. Of the guideline chapters, 9 were frequently discussed. The least frequently discussed chapter related to management of comorbid conditions. Most general practitioner (GP) participants used the AD sessions to discuss challenges with managing secondary mental conditions. In line with the protocol, opinion leaders who delivered the AD sessions largely offered evidence-based strategies aligning with the clinical guideline recommendations.The IMPRovE AD intervention component was delivered to high fidelity. The sessions adhered to the intended duration, coverage, frequency, and content allowing participating GPs to comprehend the implementation of the guideline in their own practice. This study also demonstrates that the Modified Conceptual Fidelity Framework with a mixed methods approach can support the assessment of implementation fidelity of a behavioural intervention in general practice. The findings enhance the trustworthiness of reported outcomes from IMPRovE and show that assessing fidelity is amenable for AD and should be incorporated in other studies using AD.Australian New Zealand Clinical Trials Registry ACTRN 12620001163998, November 2020.The online version contains supplementary material available at 10.1186/s43058-023-00531-2. Contributions to the literatureThis study demonstrates the value of utilising mixed methods to assess fidelity of a complex behavioural intervention.This study identifies the strengths (and limitations) of the Modified Conceptual Framework for Implementation Fidelity as a valid and comprehensive method to assessing fidelity.This study supports the use of this methodology in future studies that assess the implementation fidelity of complex behavioural interventions in general practice.Behavioural interventions aim to facilitate change in a participant’s behaviour with potential for improving health outcomes . These tThere is currently limited consensus on the key elements involved in studying intervention fidelity in randomised controlled trials, including trials of multifaceted behavioural interventions , 7–9. FoThe IMPRovE trial aimed to assess the effectiveness of a multifaceted intervention on the implementation of the ‘Clinical guideline for the diagnosis and management of work-related mental health conditions in general practice’. It investigated the effectiveness of a multifaceted intervention, comprising academic detailing (AD), enrolment in a digital community of practice and the provision of resources, on guideline adherence. . The firIn this paper, we describe the extent to which the AD component of the IMPRovE trial, was delivered according to its pre-defined process and intended function .We utilised the Conceptual Framework for Implementation Fidelity, as proposed by Carroll et al.  and modiMeasurement of adherence to the IMPRovE AD plan includes duration, frequency, coverage and content, as outlined in Table All AD sessions that were delivered within the IMPRovE intervention were audio-recorded. A minimum of 20% of the AD sessions delivered by each opinion leader were chosen for assessment, with 22.8% of all the delivered AD sessions eventually selected. This sample of AD sessions was selected through stratified random sampling, stratified by opinion leader (in order to not bias the sample to any particular opinion leaders’ adherence and to adequately represent each opinion leader who delivered the AD session) and by state. Randomisation was conducted by an independent statistician after all the AD sessions were delivered.We used a mixed method triangulation design, giving equal weight to both quantitative and qualitative data collected and analysed to provide an integrated understanding of the breadth and depth of adherence to the planned design of the AD sessions –17. A prQuantitative scoring criteria for the fidelity assessment were based on the adherence component of an adapted model of the Modified Conceptual Framework for Implementation Fidelity , 10, whiAcademic detailers were employees of the National Prescribing Service (NPS) MedicineWise, an Australian not-for-profit organisation with extensive experience in delivering educational outreach sessions to GPs on therapeutics. Their role during the AD session was to run the session and help facilitate discussion. GP opinion leaders were clinicians well-versed in compensation schemes. They were nominated by project partners, thus demonstrating their leadership amongst their peers. Opinion leaders provided a peer’s voice during the session, facilitating greater engagement from participating GPs . They haQualitative data were analysed using a conventional content analysis approach  which isAll AD sessions were scored using the fidelity coding assessment sheet  treat the person according to their situation; (ii) offer care that is within your scope of practice and use your network to provide collaborative care; and (iii) good work is good for recovery (going to work is part of the recovery).Additional File We were able to demonstrate high fidelity (89.2% on average) in the delivery of the AD component of the IMPRovE intervention using the Conceptual Framework for Implementation Fidelity, as proposed by Carroll et al.  and modiHigh fidelity has been characterised as 80–100% adherence, with less than 50% adherence representing ‘low fidelity’ of intervention delivery . InterveOur high fidelity scores were due to adequate intervention duration, the guideline being discussed frequently, as well as a good coverage of the content delivered throughout the AD sessions. There were however a number of departures from the original academic detailing protocol, and some insights of why these departures occurred are presented below.The sessions were delivered within the intended duration of 50 min ± 10 min. However, it should be noted that in the sample, three of the eight sessions had durations outside the intended range. For sessions with shorter durations, GPs being better versed and confident in guideline recommendations and thereby requiring less time to go over points may have impacted session duration. For sessions with longer durations, these factors may include more intensive discussions about challenges faced by the GPs’ practices, GPs having more questions about clinical guideline recommendations requiring more time for the opinion leader GP to answer, or opinion leaders being unprepared, which may have resulted in more administrative activities at the start of sessions.Throughout the sample of eight AD sessions, more GPs chose the case study for managing a patient with a secondary mental condition. This suggests GPs face a greater challenge or interest in the management of secondary mental health conditions. This may imply that, when educating GPs about work-related mental health conditions, more resources should be allocated towards discussing secondary mental health conditions to assist GPs with diagnosis and management of these kinds of mental health conditions.  It shoulThis study holds the view that interventions should be tailored for participants in different settings, whilst not so flexible that it compromises validity . For insAD was one component in a multifaceted behavioural intervention. There are several interacting factors that may have influenced delivery of the AD session, apart from what could be gleaned from a review of the sessions. These factors will be understood in greater depth and explained through a whole of trial process evaluation. This process evaluation for the study will be published separately.Our approach to fidelity assessment had many advantages. Firstly our data collection, through recording the zoom sessions, was unobtrusive and meant that the researchers did not influence the results. Secondly, content analysis presents a systematic procedure that is easily replicable. Thirdly, it is a highly flexible method requiring little resources to conduct. Finally, this study reinforced the value of utilising a mixed method study design to assess complex behavioural intervention fidelity, allowing us to draw on results from both quantitative and qualitative datasets to inform our understanding of AD fidelity. This research method is not common practice for assessing behavioural intervention fidelity , 23, butThere were however several limitations. Firstly content analysis can be reductive and subjective; however, this was minimised with three researchers assessing fidelity. Secondly, the sample size of eight AD sessions was small. However, we coded the recommended minimum coding of 20–40% of sessions for fidelity studies chosen from a random and stratified sample (by academic detailer and by State) across the delivery of an intervention , to adeqAs there is no consistent approach for assessing fidelity of multifaceted behavioural interventions, future research could focus on creating a consistent, validated method for assessing the fidelity of behavioural interventions in general and of AD sessions in particular. Recent modifications have been suggested for the Modified Conceptual Framework for Implementation Fidelity, such as accounting for adaptations brought by implementers or users to how an intervention was originally designed . TherefoAlthough the use of the framework to assess intervention fidelity has not fully been established, in this study , 26–28, Results from this study should give confidence that the AD portion of the IMPRovE trial intervention was delivered with high fidelity across all AD sessions. As such, there is greater confidence that the IMPRovE trial outcomes reported in the future will be the result of the AD sessions being delivered according to the protocol. High fidelity delivery also allows for a more accurate evaluation of the efficacy of this intervention on trial outcomes.Additional file 1. The IMPRovE trial.Additional file 2. Assessment sheet used for the fidelity coding.Additional file 3. The case studies of the primary mental health patient or secondary mental health patient seen.Additional file 4. Frequency with which clinical questions were discussed in the AD sessions.Additional file 5. Key messages delivered in AD sessions.


Moreover,the analysis of the supramolecular self-assembly of one of the β-turn-containingpeptide models in the solid state reveals that it forms a supramolecularhelical arrangement while maintaining the β-turn structure.The results here presented provide the basis for the use of this azepanequaternary amino acid as a strong β-turn inducer in the searchfor novel peptide-based bioactive molecules, catalysts, and biomaterials.β-Turns are one of the most common secondary structuresfoundin proteins. In the interest of developing novel β-turn inducers,a diastereopure azepane-derived quaternary amino acid has been incorporatedinto a library of simplified tetrapeptide models in order to assessthe effect of the azepane position and peptide sequence on the stabilizationof β-turns. The conformational analysis of these peptides bymolecular modeling, NMR spectroscopy, and X-ray crystallography showedthat this azepane amino acid is an effective β-turn inducerwhen incorporated at the  This quaternary amino acid displays a set ofproperties that make it especially suited for its application as peptidesecondary structure inducer. There are not many studies on the abilityof seven-membered quaternary amino acids to constrain peptide conformationson their own, i.e., without the synergistic effect of additional restrictedresidues.55 The azepane heterocycle in 1 also enablesthe incorporation of structural diversity and functionality in theconstrained peptides through the Alloc-protected nitrogen, for example,fluorescent dyes or affinity labels for visualization and immobilization.In addition, it contains two chiral centers, which can be easily definedin the synthesis step opening the possibility of studying the effectof chirality in the induction of secondary structure elements. Infact, we have reported the synthesis of the four possible diastereoisomersof the 2-oxoazepane derivative III.53 In a previous report, we have also demonstrated that theazepane 1 is an effective inducer of 310 helicesin short alanine-based pentapeptide models.56 In this article, we report the induction of single β-turnsby this quaternary amino acid in the shortest possible peptide model.We present a study of the key factors governing the folding of thepeptide chain into a β-turn, such as the position of the constrainedamino acid in the sequence and the compatibility with proteinogenicamino acids in the remaining central position of the turn.We have previously described the stereoselectivesynthesis of aquaternary azepane amino acid from commercial ornithine derivatives1.53 The 1 were first analyzed using moleculardynamics (MD) simulations with the Amber 10 suite of programs.57 Simplified tetrapeptide systems were used inwhich the N-terminal amino acid (i) was replaced by an acetyl group and the C-terminal(i + 3) by a N-methylamide. Theβ-turn induction was evaluated through its characteristic topographicparameters, i.e., H-bond distance COi–NHi+3 and the backbone dihedral angles of thecentral residues. We first studied the influence of the position ofthe constrained amino acid within the central residues of alanine-basedtetrapeptide models. As a reference, it is worth mentioning that theconformational properties of alanine-based short peptides have beenanalyzed in depth both theoretically and experimentally, showing astrong tendency for extended conformations.60 When the azepaneresidue was incorporated at the i + 1 position, molecularmodeling results showed that the 100% of the conformers within a 3kcal·mol–1 window from the global minimum stabilizetype I or III β-turns. At the i + 2 position,the global minimum also displays a β-turn, but there is anotherfamily of conformers within a 3 kcal·mol–1 windowin which the characteristic β-turn H-bond is not present (seethe Supporting Information for details).Thus, MD studies suggest that the incorporation of 1 atthe i + 1 position is preferred over that at the i + 2 position, with a higher degree of β-turn induction.This is in agreement with the preferred position for other constrainedamino acids, as the restriction in the dihedral angles imposed bythese residues at such position facilitates the β-turn nucleation.62The conformational preferencesof tetrapeptide models incorporatingthe azepane amino acid i + 1 position,we next evaluated the effect of the amino acid side chain at position i + 2, again using MD simulations. Calculations were performedin tetrapeptide models incorporating the azepane residue at the i + 1 position and the 20 proteinogenic amino acids at the i + 2 position. MD studies suggested that the azepane derivativeis a strong β-turn inducer, except when it is combined withGln, Arg, and Asp at the i + 2 position. In thesecases, less than 50% of the conformers within a 3 kcal·mol–1 window from the global minimum display a β-turnstructure. The presence of polar residues tends to destabilize theβ-turn due to competing H-bond formation between the side chainand the peptide backbone (see the Supporting Information for details). Taking these results into account, we selected a representativeset of amino acids to prepare a library of Boc-Aze(Alloc)-Xaa-NHMetetrapeptide models, where Xaa was chosen to be alanine, valine, leucine,or phenylalanine (residues with a high tendency to adopt β-turns)as well as serine, lysine, or glycine (with a medium tendency towardβ-turns). This small library covers from hydrophobic aliphaticand aromatic residues to polar ones, displaying different side chainbranching patterns. Additionally, we also prepared the correspondingalanine-based tetrapeptide model Boc-Ala-Aze(Alloc)-NHMe to experimentallyassess the influence of the azepane position in the reverse turn induction.With the azepane fixed at the 2-8 were obtained through peptide coupling betweenthe azepane residue 1 for amide protons equal to or lower than 3 ppb·K–1  are indicative of such NHs participating inan intramolecular H-bond.64 In contrast, solvent-exposedamide protons typically display values over 4 ppb·K–1, while values within 3–4 ppb·K–1 arenot conclusive. 1H NMR spectra forcompound 2 at different temperatures, as well as theplot of the chemical shift variation with temperature (Δδ/ΔT). The observed linear correlation was used to calculatethe temperature coefficient for all the amide protons. For compound 2, only NHi+3 has a temperaturecoefficient compatible with the presence of an intramolecular H-bond,suggesting the adoption of a β-turn in solution. It is worthpointing out that the splitting of the NH signal corresponding tothe i + 1 residue at the lowest temperature is likelydue to rotamers of the N-Alloc group, which coalescewhen the temperature increases.The conformational preferences in solution oftetrapeptide models 2, the temperature coefficients for all the amide protonsin derivatives 3–8 and 10 were extractedfrom VT-NMR experiments . Compounds 3 (Ala), 5 (Leu), and 7 (Ser) show the same behavior as the glycineanalog 2. Only the NHi+3 protonparticipates in an intramolecular H-bond, suggesting the stabilizationof the β-turn structure. They all contain a β-methylenegroup in the side chain, thus providing the necessary flexibilityfor the adoption of the β-turn structure. For compounds 4 , 6 (Phe), and 8 (Lys), theNHi+3 temperature coefficient falls inthe uncertainty range. Although we cannot fully conclude the formationof the β-turn in solution, the temperature coefficients forPhe (6) and Lys (8) are close to 3 ppb·K–1, suggesting some degree of β-turn folding.The valine-derived tetrapeptide model 4 clearly showsdistinct behavior, with the NHi+2 formingan intramolecular bond. This is compatible with the stabilizationof a γ-turn instead of a β-turn. γ-Turns are thesecond most common reverse turn in proteins after β-turns, formedby three consecutive residues with a H-bond between the COi and the NHi+2.65 Thus, this result demonstrates that branchingat the β-position of the side chain seems to destabilize theβ-turn conformation.Following the same protocol as that for compound 10 with the azepane residueat the i + 2 position, both NHi+2 andNHi+3 have temperature coefficients inthe uncertainty range, so there is no strong evidence of the formationof either a γ- or β-turn. Comparing the NMR data of 3 and 10, the incorporation of the constrainedamino acid at the i + 1 position seems to be morefavorable for the stabilization of the β-turn, in line withthe data obtained from the molecular modeling studies.For compound 2 and 3. N-methylamide moiety (residue i + 3) and the Boccarbonyl oxygen (residue i), characteristic of aβ-turn. Both β-turn structures are quite similar as observedwhen the two crystal structures are superimposed . In contrast, the supramolecular arrangement observedin the crystal structure of peptide derivative 3 belongsto the tetragonal system. It forms supramolecular right-handed helicalassemblies with small internal pores  and COi+1 of moleculeA and the NHi+1 and NHi+2 of molecule B, respectively  seems to largelydisrupt the β-turn. We have also paid attention to the supramolecularself-assembly behavior of two β-turn-containing peptide modelsin the solid state. When alanine is incorporated at the i + 2 residue, the tetrapeptide model self-assembles into a supramolecularright-handed helical structure, stabilized by a defined network ofintermolecular H-bonds along with the characteristic β-turnintramolecular H-bond. The results here presented therefore providethe basis for the use of azepane quaternary amino acids as effectiveβ-turn inducers with widespread potential applications in thedevelopment of novel bioactive molecules, catalysts, and biomaterials.The stabilization of the peptide secondarystructure elements isof interest to achieve a fundamental understanding of complex protein–proteininteractions in the search of novel therapeutic agents. In particular,β-turns are some of the most common secondary structures foundin proteins. They play a key role in protein folding and stabilizationas well as in protein–protein and peptide–protein recognition.Moreover, they are an attractive scaffold with relevant applicationsin peptide-based catalysis and in supramolecular materials. The useof constrained quaternary amino acids is one of the most common strategiesused to induce reverse turns in short peptide sequences. In this context,we have previously reported the stereoselective synthesis of a novelazepane-derived quaternary amino acid.1 in dry THF (9 mL) was treated with H-Gly-NHMe·TFA , (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, and triethylamine .After the mixture was stirred at room temperature for two days, thesolvent was evaporated to dryness. The crude was dissolved in EtOAcand washed successively with 10% aq. soln. citric acid (2×),10% aq. soln. NaHCO3 (2×), H2O (1×),and brine (1×). The organic phase was dried over MgSO4 and evaporated to dryness. The residue was purified by centrifugalthin-layer chromatography using a gradient from 200:1 to 10:1 of MeOH/CH2Cl2, yielding 2 as a white, amorphoussolid . HPLC: tR = 6.26min (gradient from 15% to 95% CH3CN/0.1% formic acid inH2O/0.1% formic acid over 10 min). 1H NMR : δ 7.96 , 7.44 , 6.95 , 5.93,5.27 ,5.18 ,4.53 , 3.70 , 3.52–3.56 , 3.35 , 3.09 , 2.58 , 2.27 , 2.22, 1.69 , 1.65 , 1.51 ,1.43 , 0.82 . 13C{1H} NMR :δ 173.6 and 169.3 (CONH), 155.3 , 155.6 , 155.1 , 133.5 , 116.4 , 78.7 , 64.8 , 63.4 (4-C), 47.2, 46.9 , 45.6 , 45.3 , 42.4 (α-C),39.1 (3-C), 30.2 , 30.5 , 28.0 , 25.1 (N–CH3), 20.6 , 20.1 ,13.0 (3-CH3). MS (ES+): m/z = 427.19 [M + H]+, 853.43 [2M+H]+. Elementalanalysis: calcd (%) for C20H34N4O6 C 56.32, H 8.04, N, 13.14. Found (%): C 56.25, H 8.08, N13.19.A solutionof the azepane-derived amino acid 1 in dry CH2Cl2 (7 mL) was treated with (benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate , H-Ala-NHMe·HCl, and triethylamine . Afterthe reaction mixture was stirred at room temperature for 15 h, thesolution was diluted with EtOAc and washed successively with 10% aq.soln. citric acid (2×), 10% aq. soln. NaHCO3 (2×),H2O (1×), and brine (1×). The organic phase wasdried over Na2SO4 and evaporated to dryness.The residue was purified on a silica gel column using EtOAc/hexane(8:1) as solvent, yielding 3 as a white, amorphous solid. HPLC: tR = 10.99 min (gradientfrom 5% to 80% CH3CN/0.05% TFA in H2O/0.05%TFA over 20 min). Caution! The use of BOP as couplingreagent is discouraged because of the formation of carcinogenic HMPA,with PyBOP being a convenient alternative. 1H NMR : δ 7.83 , 7.80 , 7.62 , 7.00 , 6.94 , 5.92 ,5.26 ,5.18 ,4.52 , 4.24 , 4.23 , 3.58 , 3.45 ,3.29 , 3.11 , 3.01 ,2.56 , 2.30 , 2.07 , 1.68 , 1.63 ,1.46 , 1.42 , 1.21 , 0.82 , 0.80 . 13C{1H} NMR: δ 173.1 and 172.5 (CONH), 155.8 and 155.4, 155.7 and 155.2 ,133.6 , 116.7 , 116.5, 78.9 , 78.8 , 65.0, 63.7 (4-C), 48.2 , 47.7 , 47.1 , 45.5 , 45.3 , 40.8 (3-C), 30.0(5-C), 28.2 , 25.5 (NCH3), 20.8 , 20.1 , 17.6 , 13.3 , 13.2 . MS (ES+): m/z = 441.2 [M + H]+, 463.3 [M + Na]+, 881.7 [2M+H]+, 903.5 [2M+Na]+. Elementalanalysis: calcd (%) for C21H36N4O6 C 57.25, H 8.24, N 12.72. Found (%): C 57.08, H 8.07, N 12.80.A solutionof the azepane-derived amino acid 2–8 and 10 at 7–10mM concentration in DMSO-d6 were preparedand transferred to an NMR tube. 1H NMR spectra were acquiredat different temperatures (25–85 and 10 °C steps). Thevariation of the amide proton chemical shifts was plotted against the variation of temperature , and the data fitted to a straight line. The slope of the equationrepresents the temperature coefficient .Solutions of compounds 2 (1.5 mg) was dissolved in MeOH (1 mL) andthe mixture was put in a crystallizing dish, resulting in spontaneouscrystallization after 12 days at 4 °C in a closed jar. X-raydiffraction was performed at the BL13-XALOC beamline at ALBA Synchrotronwith the collaboration of ALBA staff.Purecompound 3 (5 mg) was dissolved in MeOH (5 mL)and the mixture was put in a crystallizing dish, resulting in spontaneouscrystallization after 20 days at 4 °C in a closed jar. X-raydiffraction was performed in a Bruker MicroStar 2.7 kW, with a four-circlegoniometer, with κ-geometry, and Bruker CCD detector, usingCuKα radiation.Pure compound


A graphene/poly(methyl methacrylate) (PMMA) closed cavity resonator with a resonant frequency at around 160 kHz has been fabricated. A six-layer graphene structure with a 450 nm PMMA laminated layer has been dry-transferred onto the closed cavity with an air gap of 105  Graphene has aroused much attention from the research and industrial community since it was discovered  due to iThe unique mechanical and electrical properties of graphene also show that it is an interesting material for ultrasonic sensing. The large Young’s modulus of graphene suggests that graphene-based membranes can be easily designed to reach a high resonant frequency, typically in the range of megahertz ,21,24,25To date, there has been limited study of graphene-based resonators reported to cover the ultrasonic frequency range between 20 kHz and 1 MHz. To achieve a relatively lower ultrasonic frequency range (below 1MHz), a larger size of graphene-base membranes is required, which increases the complexity of graphene transfer and the difficulty in suspending the graphene-based membrane over the substrate without collapse. To address these two problems, an ultrasonic transducer [In this work, a fully clamped graphene/PMMA closed cavity resonator at a resonant frequency less than 200 kHz will be presented. To avoid the membrane being transferred on the supporting ring and assembled onto the substrate afterwards, a one-step graphene dry transfer process has been developed by our group . A six-lt and R is the radius of the membrane; An optical image of the graphene/PMMA ultrasonic closed cavity resonator is shown in The fabrication process of the graphene/PMMA closed cavity ultrasonic sensor is shown in lication . In thisThe graphene/PMMA ultrasonic resonator has been actuated mechanically, electro-statically and electro-thermally to characterize its dynamic behavior. For the mechanical actuation, the graphene/PMMA ultrasonic resonator has been placed and attached to the piezoelectric disk. By applying a voltage to the piezoelectric disk, ultrasound vibration has been generated and actuated the substrate of the resonator. For the electro-static actuation, silver paste has been attached to the graphene layer to work as the top electrode. The patterns etched into the silicon with the resistivity of 1–10 For mechanical actuation, the varying AC voltage from 0.2 V to 3 V and a constant 1 V DC voltage with a frequency sweep from 150 kHz to 220 kHz have been applied to the piezo-disk. The frequency response of the membrane is shown in For electro-static actuation, the voltage of constant 1 V DC and varying AC voltage from 4 V and 9 V with a frequency sweep between 120 kHz to 200 kHz have been applied between the membrane and substrate. The frequency response of the graphene/PMMA membrane is shown in For electro-thermal actuation, the frequency response of the resonator actuated by increasing 1 V to 9 V AC and 1 V DC voltage applied to the silver paste on the graphene/PMMA membrane with the frequency range from 140 kHz to 220 kHz is illustrated in The vibration amplitude of the graphene/PMMA membrane over the closed cavity is shown in The dynamic behavior of the graphene/PMMA closed cavity ultrasonic sensor is summarized in oftening ,33,34 anoftening .In mechanical and electro-static actuation, the change in frequency shift and quality factor versus the input signal has been detected to be relatively small compared to the electro-thermal case, as shown in In the case of the electro-thermal actuation, the change in quality factor can be temperature-related. The frequency shift at resonance is evident in the frequency response c. The reAs shown in The change in the quality factor has been studied in the resonator under the electro-thermal actuation. The quality factor has been measured to increase from around 36 to 40 when the AC voltage rises from 2 V and 3 V. A decrease in the quality factor has been observed when the AC voltage changes from 3 V to 8 V. A small increase in the quality factor was measured when the AC voltage increased from 8 V to 9 V, as shown in The mode shapes at the resonant frequencies obtained by different actuation methods are shown in The overall tension and strain can be derived from Equation  and the This is the first time that a graphene-based closed cavity ultrasonic resonator has been fabricated and actuated in the atmosphere successfully. Using the graphene dry transfer method with Kapton tape as the supporting frame, developed by our group, a graphene/PMMA closed cavity sensor at a resonant frequency of around 160 kHz has been fabricated. The graphene/PMMA closed cavity resonator has been actuated mechanically, electro-statically and electro-thermally. The amplitude of the membrane has been observed to be linear with AC voltage for the mechanical and electro-static actuation and quadratic with AC voltage for the electro-thermal actuation. The membrane has been observed to exhibit a  mode at the resonant frequencies. The membrane can be tuned by up to 4% by varying the AC voltage via the electrodes connected to the graphene/PMMA membrane, and a nonlinear frequency shift under electro-thermal actuation has been detected. The strain in the membrane under the three actuation methods has been estimated to be around 0.08%. The device shows the possibility of applying graphene as an ultrasonic detector and opens a door to the fabrication of graphene-based ultrasonic sensors at a lower ultrasonic frequency of less than 200 kHz.