Spider silk is one of the hottest biomaterials researched currently, due to its excellent mechanical properties. This work reports a novel humidity sensing platform based on a spider silk-modified quartz tuning fork (SSM-QTF). Since spider silk is a kind of natural moisture-sensitive material, it does not demand additional sensitization. Quartz-enhanced conductance spectroscopy (QECS) was combined with the SSM-QTF to access humidity sensing sensitively. The results indicate that the resonance frequency of the SSM-QTF decreased monotonously with the ambient humidity. The detection sensitivity of the proposed SSM-QTF sensor was 12.7 ppm at 1 min. The SSM-QTF sensor showed good linearity of ~0.99. Using this sensor, we successfully measured the humidity of disposable medical masks for different periods of wearing time. The results showed that even a 20 min wearing time can lead to a >70% humidity in the mask enclosed space. It is suggested that a disposable medical mask should be changed <2 h. Spider silk is well known for its outstanding mechanical properties. One of them is that spider silk is particularly sensitive to water . When weQuartz tuning forks (QTFs) have been traditionally used as a timing reference in wristwatches. The QTF is a bimorph cantilever based on the piezoelectric properties of the quartz . The senIn this work, a sensitive and cost-effective humidity sensor was developed by using quartz-enhanced conductance spectroscopy (QECS) . CompareQTFs were purchased from the Shenzhen XinChuangYue Electronic components Factory, with a resonance frequency of ~32.7 kHz and a Q factor of >60,000 in a sealed vacuum metallic package and ~10,000 in the air after removal of the metallic housing. The geometrical parameters were measured by an optical stereomicroscope. The prong length, prong width, and prong spacing of a QTF were 3.8 mm, 0.6 mm, and 0.3 mm, respectively. The effective spring constant of the QTF is ~20 kPa . The effTo evaluate the humidity sensing performance of the SSM-QTF, we employed the experimental setup shown in f of the QTF can be expressed as [QTFk, QTFm, and QTFE are the spring constant, effective mass, and Young’s modulus, respectively. The t, l, and w are the QTF prong thickness, QTF prong length, and QTF prong width, respectively. We considered that the mass of the spider silk was negligible, i.e., QTFdm≪QTFm. The resonance frequency shift, after modification by the spider silk, can be expressed as:The resonance frequency essed as ,51:(1)fQQTFdk value, which results from the spider silk spring constant ksilk. Due to the fact that the QTF and the spider silk were connected in parallel, the effective spring constant effk of the SSM-QTF can be expressed as:The resonance frequency shift can be attributed to the The electrical parameters of the SSM-QTF were measured by the abovementioned system in f = 241.3 Hz. This can be attributed to the mass increase caused by the spider silk and the glue.A bare QTF without modification was also evaluated, under the same experimental conditions. The frequencies of the sine signals were scanned from 32,500 Hz to 32,560 Hz and from 32,725 Hz to 32,805 Hz with a step of 0.3 Hz for SSM-QTF and standard QTF, respectively. The resonance curves of the bare QTF and SSM-QTF are shown in f = 25.95 Hz. This can be attributed to the modulus of spider silk being inversely proportional to the humidity; the effective modulus of the SSM-QTF decreased with the increase in the humidity [f was caused by the spider silk, not the QTF itself.humidity . The curQ factors of the SSM-QTF with different humidities. As the humidity increased, the spider silk absorbs water molecules, increasing the additional mass attached to the QTF. In addition, as the humidity increased, the damping effect by the water absorption into spider silk increased; thus, the quality factor of the SSM-QTF decreased [Q factor decreased from 5253 to 2790, by >2400. For comparison, the Q factor change of a bare QTF without spider silk modification was <200, shown in the inset of ecreased . As a reThe relative humidity (RH) was converted into absolute humidity by the Vaisala humidity calculator. With an absolute humidity change from 8042 ppm to 24,056 ppm, the detection sensitivity of the SSM-QTF was calculated to be ~12.7 ppm. This humidity dynamic range was limited by our gas stream system, which can be improved by a better humidifier. The comparison of the SSM-QTF with current technology such as optical fibers, cantilever, and string resonators are summarized in −3 Hz at the optimum time of ∼30 min. With the optimal integration time, the detection sensitivity can be improved to ~0.8 ppm.The resonance frequency of the SSM-QTF was measured with a fixed relative humidity. The humidity in the gas chamber was well controlled by using a mass flow controller  with a relatively low flow rate but higher resolution. With a relative humidity of 20 ± 0.1% RH, a resonance frequency of 32,542.88 Hz was obtained. Since the relative humidity is sensitive to temperature, the ambient temperature condition has to be properly maintained throughout the experiment. For the tested humidity range, the temperature in the humidity chamber has been monitored to be ~24 °C. It can be seen from The COVID-19 pandemic has forced the global population to adopt new ways of living, including the possible use of masks. Research has been conducted to evaluate the potential consequences of wearing masks for long periods ,67. AmonIn the measurement, the humidity inside a disposable medical mask made of polypropylene was evaluated. The disposable medical masks were worn by the author Leqing Lin for different periods; then, they were taken off quickly to cover the SSM-QTF sensor in a sealed space. The humidity of the covered air inside the medical mask was then measured. For each measurement, the medical mask was replaced by a new one. The time clock was restarted. Except for the wearing time, all the other conditions were the same in the repeated experiments. The enclosed spaces were set up to prevent humidity interference from the outside atmosphere. Resonance frequencies of the different wearing time of the masks were recorded by a LabVIEW program. The obtained results were shown in This work featured a novel humidity sensing platform based on a quartz tuning fork (QTF) with a section of natural spider silk. The experimental results showed that the higher the environmental humidity, the lower the resonance frequency of the spider silk-modified QTF. The variation of the resonance frequency of the QTF modified by spider silk was closely correlated with the change in the environmental humidity. The evaluation was conducted in relative humidity from ~25% RH to ~80% RH. The resonance curves were recorded to obtain the SSM-QTF frequency variations by Lorentz fitting. With a 1 min working time, the detection sensitivity of ~12.7 ppm was obtained.As proof of concept, the humidities of disposable medical masks, worn for different periods, were evaluated. The results suggested that the masks should be changed every 2 h due to excessive humidity, which may cause potential facial skin discomfort. Currently, the acquisition time for the resonance frequency and Q factor requires ~1 min. This time can be shortened by narrowing the frequency scanning time and range. Allan deviation was measured over a long time with a fixed humidity. With 30 points of integration, the detection sensitivity can be improved to ~0.8 ppm. Future work will include the development of a wearable respiration sensor to measure the humidity inside a medical mask. Compared to the current electrochemistry sensor ,76, the


An acceptable recovery has been revealed in the real sample analysis. The electrochemical sensing behaviour of the composite indicates that it may be a promising candidate for a glucose sensor and it significantly extends the range of applications in the electrochemical field.In this work, a novel electrochemical sensor was developed by electron-withdrawing substituent modification of 1-phenyl-3-methyl-4-(4-fluorobenzoyl)-5-pyrazolone on a graphene-modified glassy carbon electrode (HPMpFP-graphene/GCE) for glucose detection. The results of characterizations using a scanning electron microscope, Fourier transform infrared spectroscopy, Raman spectroscopy, and nuclear magnetic resonance spectroscopy showed the successful fabrication of HPMpFP-graphene nanocomposite, which served as an electroactive probe for glucose detection. The electron transfer ability of HPMpFBP-graphene/GCE has been successfully revealed using cyclic voltammetry and electrochemical impedance spectroscopy results. The good electrochemical performance was shown by well-defined peak currents of square wave voltammetry under various parameters, including pH, HPMpFP and graphene composition, and scan rate effect. A high electrochemically evaluated surface area using chronoamperometry suggested that the present glucose detection response was intensified. The chronoamperometry results at a work potential of 0.4 V presented a wide linear range of 1 × 10 Clark and Lyons proposed the use of glucose oxidase entrapped inside a semipermeable dialysis membrane built on an oxygen electrode almost 50 years ago . After tFood composition information, including glucose content, is vital for the modern food industry as low-quality foods are associated with increased morbidity, mortality, human suffering, and economic burden . Hence, Numerous articles described the use of the enzymatic electrochemical sensing technique for glucose detection in food and beverage samples –9. Many 2 carbon atoms organized in a honeycomb-like form. Graphene has 260 times the surface area of graphite and twice that of a carbon nanotube, making it the most densely packed material known ). As shown in Ep=Epc − Epa) of 0.367 V. The redox peak potentials were raised by 0.158 V and 0.139 V with the addition of graphene and HPMpFBP-graphene composite. For bare GCE, the current ratio of the oxidation peaks to the corresponding reduction peaks (ipa/ipc) was 1.04. For graphene/GCE, the current ratio was 0.93, and for HPMpFP-graphene/GCE, the current ratio was 0.97. As the ratios were close to one, these results were due to a reversible voltammogram (ipa/ipc = 1)  with 1.0 M KCl. The direct part of the semicircle depicts the diffusion process, while the whole semicircle represents an active control barrier that acts to reduce the flow of electrons at the electrode contact, which is also known as electron transfer resistance (Rct) [3 [Fe(CN)6] at the bare GCE showed a small semicircle in the high-frequency area and is a straight line in the low-frequency region, as shown in Rct. The Randles equal circuit viable with the impedance spectrum is shown in the inset of Rs, Cdl, and Zw.The EIS was carried out to take into consideration the electrochemical characteristics of the modified electrodes via the use of a redox probe of 4 mM Kce (Rct) . The EISRct for HPMpFBP-graphene/GCE, which was found to be lower (20.86) than graphene/GCE (43.14) and bare GCE (80.97). The electrode with the lowest Rct value has the best conductivity. It tends to be summed up that the arrangement of Rct on the electrodes expanded in the request for HPMpFP-graphene/GCE < graphene/GCE < unmodified GCE. The astounding conductivity of HPMpFP-graphene/GCE demonstrates the extraordinary electron move capacity. In the development of electrochemical sensors, this is an important factor to consider. The electron move rate steady (kapp) values obtained from Equation (3 [Fe(CN)6] undergoes a redox reaction, the amount of moles of electrons transported (n) is equal to one.Using the Randles equivalent electrical circuit, it was possible to calculate the value of e/GCE 43.4 and barkapp value achieved for HPMpFP-graphene/GCE was greater than the corresponding values found for graphene/GCE and bare GCE. Consequently, it can be concluded that the HPMpFP-graphene/GCE, which benefited from the synergistic impact of the HPMpFP complex and the graphene composite, has produced excellent electrochemical sensor characteristics as a result of its use.From The electrochemical effectiveness of various prepared electrodes was determined using CV with the presence and absence of 0.1 mM glucose, which was tested in 0.1 M PBS (pH 7) electrolyte solution. The experiments shown in π-π strength and hydrogen bonding to form the composite, but it is also used to construct a conductive interconnivance network that can prevent graphene coagulation and increase surface area. Third, introducing electron-withdrawing substituents such as fluorine into the 1,3-β-diketone position of the HPMpFP may improve the ligand's electrical characteristics, thereby enhancing glucose detection at the modified electrode surface.This modified electrode's electrocatalytic reaction to glucose is beneficial for the following reasons. First, the outstanding electrical conductivity of graphene helps increase the glucose-attracting ability of the resulting electrode, thereby increasing its large surface area. Second, graphene is not only integrated with HPMpFP aromaticity that is adsorbed on graphene surfaces through Ep) and pH levels is shown. It is possible to describe this connection mathematically using the linear regression equation, which may be expressed as follows:Using SWV, the impact of pH (6.8 to 7.6) on the peak glucose potential at the HPMpFP-graphene/GCE in 0.1 M PBS was investigated . The resThe slope was neared to the theoretical value of 58.6 mV/pH. This indicates that during the glucose redox process the total number of electrons and protons taking part was identical . FollowiµL resulted in a substantial rise in peak current, while increasing the dispersion volume further to 2.0 µL resulted in a considerable decrease in peak current. This is likely to become more difficult due to the excessive thickness of sample dispersion and electrode surface diffusion. Therefore, 1.0 μL of HPMpFP-graphene composite dispersion was selected for electrode preparation in this study.A droplet of HPMpFP-graphene composite was cast on the GCE surface based on the electrode preparation stage, and a thin layer of the sample was left behind on the GCE due to solvent evaporation. Therefore, by changing the amount of dispersion cast on the GCE surface, the thickness of the HPMpFP-graphene composite layer could be altered. The SWVs of prepared electrodes with different quantities of composite HPMpFP-graphene cast on GCE are shown in Epa) and cathodic peak (Epc) potentials shifted positive and negative, respectively. The values of peak separation (ΔEp = Epc–Epa) were calculated to be between 0.215 and 0.455 V at different scan rates, which illustrates the sluggish electron transport kinetics. The difference between the anodic and cathodic peaks in these redox reactions is related to ion transport resistance in these processes  is 7.6 × 10−6 cm2/s, and the electron number is one [The surface area of the working electrode is indicated by r is one .Q and t1/2 was used to derive the following equation for unmodified GCE, graphene/GCE, and HPMpFP-graphene/GCE, which was found to follow with the findings presented in Figure 8(a) for all three electrodes.The slope of the linear connection between −3 mC/s1/2 (bare GCE), 1.126 × 10−3 mC/s1/2 (graphene/GCE), and 2.365 × 10−3 mC/s1/2 (HPMpFP-graphene/GCE), A was calculated to be 1.12 × 10−4 cm2, 1.01 × 10−3 cm2, and 6.13 × 10−3 cm2 for unmodified GCE, graphene/GCE, and HPMpFP-graphene/GCE, respectively. In addition to increasing the number of electrode reaction sites, increasing the adsorption capacity, and intensifying the current response of glucose, the increased electrode surface may also improve the sensitivity and detection limit of this sensor.According to the slopes of 0.124 × 10Q was plotted against time t1/2 ), it revealed a linear connection with the following equation, where the slope was 1.471 × 10−3 mC/s1/2 and the intercept (Qads) was 0.098 × 10−3 mC.In addition, HPMpFP-graphene/GCE was also subjected to chronocoulometry analysis in the absence and presence of 0.1 mM glucose for 30 minutes. When Q and t1/2, the intercept of the linear relationship corresponds to Qads. As n = 2, A = 5.31 × 10−4 cm2, and C = 0.1 mM, the D was estimated to be 1.6194 × 10−4 cm2/s. The adsorption capacity (Γs) of glucose at the HPMpFP-graphene/GCE was calculated to be 9.58 × 10−7 mol cm−2, following with equation:When the background is subtracted from the linear connection between The dependence of the potential applied to the amperometric signal of HPMpFBP-graphene/GCE is shown in µM. As shown in µM, then 15 µM, and finally 25 µM, before being stopped. Later, the increments were raised by 45 µM at a time, until the concentration reached 90 µM. Finally, 100 µM increments were made until the final 1000 µM step-in addition.Rather than using SWV or CV, the chronoamperometry approach was utilized in this research for quantitative glucose analysis since it has better sensitivity and resolution than other methods. After the first administration, a 50-second interval was given among the following doses, and the glucose concentration was varied from 5 to 1000 μM. There were two linear regions in this range (μA) = 0.07 + 1.46 C (R2 = 0.996) with a sensitivity of 11.42 μA/mM/cm2. Meanwhile, the current continued to rise in the second area, starting at 88 µM, when the linear regression equation was found to be I (μA) = 0.032 + 6.074 C (R2 = 0.998) and the sensitivity was measured as 5.22 μA/mM/cm2. It was estimated that the detection limit was 0.74 μM (S/N = 3). Compared with other carbon-based materials reported in the literature . The reproducibility was tested using six identically produced electrodes, and the relative standard deviation (RSD) was determined to be 2.52 ± 0.51% . Ten conThe stability of the as-prepared sensor was determined by monitoring the current response to glucose detection after two weeks of operation . The curt-test analysis to determine whether any of the findings obtained in the presence of a known amount of glucose added to the PBS solution were legitimate. As indicated in p < 0.05, there was no statistically significant difference, while for p > 0.05, there was a statistically significant difference. These findings suggest that nonenzymatic HPMpFP-graphene composite sensors may be employed in glucose determination in beverages.To show the viability of nonenzymatic HPMpFP-graphene/GCE measurements in real-world glucose, this precise sensor was used to detect glucose concentrations in orange juice and milk samples. In summary, the fabrication of HPMpFP-graphene/GCE sensors for selective glucose detection was described in this study. Under optimal conditions, the peak current for glucose detection on HPMpFP-graphene/GCE was significantly higher than the peak currents on graphene/GCE and unmodified GCE. As a result, the integration of the greater surface area and unique graphene conductivity of the composite with the HPMpFP complex significantly improved the performance of the sensor. Diffusion-controlled redox processes of glucose were shown to be more common in the presence of HPMpFP-graphene/GCE. Low detection limits for glucose and a wide concentration range for glucose were shown by the HPMpFP-graphene/GCE, demonstrating good selectivity and sensitivity towards glucose. The HPMpFP-graphene/GCE was effectively utilized to detect glucose in certain orange juice and milk samples, with a recovery rate ranging from 90 to 103%.


Caenorhabditis elegans. We demonstrate that TDP-43 specifically enhances tau but not Aβ neurotoxicity, resulting in neuronal dysfunction, pathological tau accumulation and selective neurodegeneration. Furthermore, we find that synergism between tau and TDP-43 is rescued by loss-of-function of the robust tau modifier sut-2. Our results implicate enhanced tau neurotoxicity as the primary driver underlying worsened clinical and neuropathological phenotypes in AD with TDP-43 pathology, and identify cell-type specific sensitivities to co-morbid tau and TDP-43. Determining the relationship between co-morbid TDP-43 and tau is crucial to understand, and ultimately treat, mixed pathology AD.Although amyloid β (Aβ) and tau aggregates define the neuropathology of Alzheimer's disease (AD), TDP-43 has recently emerged as a co-morbid pathology in more than half of patients with AD. Individuals with concomitant Aβ, tau and TDP-43 pathology experience accelerated cognitive decline and worsened brain atrophy, but the molecular mechanisms of TDP-43 neurotoxicity in AD are unknown. Synergistic interactions among Aβ, tau and TDP-43 may be responsible for worsened disease outcomes. To study the biology underlying this process, we have developed new models of protein co-morbidity using the simple animal  Summary: TDP-43 co-pathology is a frequent feature of Alzheimer's disease. This paper shows that TDP-43 specifically enhances tau neurotoxicity, but not amyloid β, to promote pathological protein accumulation, behavioral impairment and selective neurodegeneration. Both Aβ its own . Recent ysiology .TARDBP, are causative for ALS, demonstrating that dysfunctional TDP-43 actively contributes to neurodegenerative pathways  and ∼50% of cases of frontotemporal lobar degeneration (FTLD-TDP) . TDP-43 pathways .Following its identification as a key pathologic protein in ALS and FTLD-TDP, TDP-43 pathology was also found to occur as a secondary pathology in other neurodegenerative diseases . In partin vitro or in vivo. In cell and mouse models, TDP-43 regulates mRNA splicing of tau exon 10, shifting the ratio of tau microtubule binding repeats from the normal balanced ratio of 3R/4R-tau to a higher proportion of 4R-tau . Expression of TDP-43 in this strain is maintained at very low levels by the insertion of a self-cleaving ribozyme between the TDP-43 coding sequence and the 3′ untranslated region (UTR) of the transgene, which results in the inactivation of most, but not all, transgene-derived transcripts   . In ordeC. elegans models of neurodegenerative disease (Movies 1-8). In contrast, Aβ+TDP Tg-low animals exhibited no change in motility from control strains .Visually, the tau+TDP Tg-low animals exhibit dramatically worse motility than either tau Tg or TDP Tg-low strains, manifested by increased uncoordination in their movement. In fact, impaired motility has been used successfully as a readout for neuronal dysfunction or neurodegeneration in other  disease  and repr strains C,D. To c strains . SimilarAs controls, we examined whether tau Tg or TDP Tg-low would synergize with a non-AD related neurodegenerative disease model expressing 86 repeats of polyglutamine pan-neuronally (polyQ Tg) . PolyQ TWe next examined several well-characterized behavioral outputs in the tau+TDP Tg-low model. Mechanosensation integrates signaling from dopaminergic, glutamatergic and serotonergic neurotransmitter classes of neurons to generate a stereotypical response to light touch. To evaluate this, we employed an assay evaluating responses to gentle touch with an eyelash hair ten times, alternating head and tail touches. Consistent with previous reports , tau Tg Fig. S3). Therefore, TDP-43 potentiates pathological tau protein changes, which may be the primary driver of neurotoxicity in co-expression models of tau and TDP-43.It is possible that the motility and sensory impairments observed in tau+TDP Tg-low are due to accumulation of neurotoxic pathological tau or TDP-43. To determine whether the co-expression of tau and very low levels of TDP-43 influenced their protein accumulation, we measured protein levels in tau+TDP Tg-low by immunoblotting. We found that both total and phosphorylated tau increased in tau+TDP Tg-low A-C, but,Fig. S4). Conversely, all transgenic strains surveyed had a significant decrease in glutamatergic neurons compared with non-Tg animals at day 1 of adulthood; however, by day 4 of adulthood, both tau Tg and tau+TDP Tg-low animals had lost significantly more neurons than non-Tg or TDP Tg-low animals alone . We also evaluated whether aging differentially affected these various neurotransmitter classes of neurons by assessing neurodegeneration over time at day 1 and day 4 of adulthood. This survey revealed distinct differences in neuron vulnerabilities by neurotransmitter class to co-morbid tau and TDP-43, and to aging. We found no significant difference in dopaminergic neurons at either day 1 or day 4 of adulthood between all strains  (. We found sut-2 null mutations [sut-2 (−)] robustly protected against tau+TDP Tg-low uncoordinated motility (sut-2(−) could reduce the accumulation of tau or TDP-43 protein. We found that sut-2(−) reduced both total tau protein and pathological phosphorylated tau in tau+TDP Tg-low animals (sut-2(−) did not significantly reduce levels of total or phosphorylated TDP-43 in tau+TDP Tg-low  . We founmotility A and GABmotility B,C. We t animals A-C. HoweP Tg-low A,D,E. TaC. elegans model of combined tau and TDP-43 proteotoxicity, we demonstrate that the presence of TDP-43 promotes the accumulation and hyperphosphorylation of tau, whereas tau does not have the same effect on TDP-43 protein. We also show that the combination of tau and TDP-43 differentially affects neurons of different neurotransmitter classes, indicating some degree of selective cell-type vulnerability. Further, we show that suppression of tau neurotoxicity using the genetic modifier sut-2 protects against effects of tau and TDP-43 synergism.Through a series of functional experiments in a C. elegans TDP-43 homolog, tdp-1, impacted neurotoxicity of tau Tg animals, but did not see any difference in tau-mediated motility dysfunction . The C. elegans tdp-1 gene lacks the C-terminal G-rich domain present in human TDP-43, which is the site of pathological phosphorylation, the location of most ALS-causing mutations in the TARDBP gene, and comprises the fibrillar core of aggregates in FTLD-TDP  . Notably, it has been previously shown that tau synergizes with Aβ in d Aβ Tg) . We alsoin vitro TDP-43 does not modulate the expression of APP, nor does APP affect TDP-43 expression  only modestly decreases TDP-43 protein accumulation and does not protect against TDP Tg motility impairment. The near complete amelioration of motility defects and neurodegeneration in the combined tau+TDP-low Tg model in the presence of sut-2(−) supports the concept that enhanced tau neurotoxicity drives the exacerbated phenotypes. However, the mechanism of this protection remains unknown. SUT-2 is nuclear localized, binds mRNA and regulates poly-A tail length  was maintained as previously described (bkIs1441[Paex-3::Tau WT(4R1N)+Pmyo-2::dsRED] (gnaIs2[Pmyo-2::YFP+Punc-119::Abeta1-42] (dvIs50[pCL45(snb-1::Abeta1-42::3′UTR(long))+mlt-2::GFP] (bkIs241[pF25B5.3::Q86-YFP] (oxIs12[Punc-47::GFP + lin-15(+)] +Pmyo-2::dsRED]; bkIs1943[Psnb-1::hTDP-43 WT::K4aptazyme::unc-54 3′UTR+Pmyo-3::mCherry] was generated by crossing CK1441 and CK1943. The CRISPR-generated whole-gene deletion CK3011 sut-2(bk3011) was generated as described in sut-2(bk3011) to generate NLS23 sut-2(bk3011); bkIs1441[Paex-3::Tau WT(4R1N)+Pmyo-2::dsRED]; bkIs1943[Psnb-1::hTDP-43 WT::K4aptazyme::unc-54 3′UTR+Pmyo-3::mCherry], NLS24 sut-2(bk3011); bkIs1943[Psnb-1::hTDP-43 WT::K4aptazyme::unc-54 3′UTR+Pmyo-3::mCherry] and NLS25 sut-2(bk3011); bkIs1441[Paex-3::Tau WT(4R1N)+Pmyo-2::dsRED]. Strains with GFP-marked serotonergic neurons were generated by crossing the reporter strain JPS617 [Ptph-1::GFP] with CK1441, CK1943 and NLS19. Strains with GFP marked dopaminergic neurons were generated by crossing the reporter strain WG1 [Pdat-1::GFP] with CK1441, CK1943 and NLS19. Strains with GFP marked glutamatergic neurons were generated by crossing the reporter strain OH10972 [Peat-4::GFP] with CK1441, CK1943 and NLS19. Strains with GFP-marked cholinergic neurons were generated by crossing the reporter strain LX929 [Punc-17::GFP] with CK1441, CK1943 and NLS19. Strains with GFP-marked GABA-ergic motor neurons were generated by crossing sut-2(bk3011) with CK1441, CK1943, NLS19 and the reporter strain EG1285 [Punc-47::GFP]. See Table S1 for complete list and descriptions of all strains used in this study.Wild-type escribed . Previou::dsRED] , GRU102 eta1-42] , CL2355 -2::GFP] , CK241 bQ86-YFP]  and EG12n-15(+)] . CK1943 C. elegans were transferred to 35 mm NGM assay plates seeded with 20 μl OP-50 bacteria. Animals were allowed to acclimate to conditions on assay plates at room temperature for at least 30 min before recording movements for 1 min at 7.5 frames per second. For thrashing assays, staged-matched day 1 adult C. elegans were given 30 min to acclimate to the assay room conditions. Approximately 50 animals were then transferred to a 35 mm assay plate by way of washing in 1 ml M9 buffer, given 1 min to standardize swimming behavior, followed by a 1 min recording time at 14 frames per second. Tracks were verified and repaired as needed. Figures show results from at least three independent replicates.Unstimulated activity and thrashing behaviors were assessed using the WormLab system (MBF Bioscience). For unstimulated activity assays, stage-matched day 1 adult Mechanosensation assays were adapted from EPG comparisons were performed using the NemaMetrix ScreenChip System and associated software packages NemAquire and NemAnalysis, and Microsoft Excel. Stage-matched day 1 adult animals were washed in M9 and then incubated at room temperature in 10 mM Serotonin in M9 buffer for 20 min to induce pharyngeal pumping. Animals were loaded into a ScreenChip20 cartridge under vacuum. The electrical signal  was recorded individually for 2 min. Average pump frequency and duration readouts from NemAnalysis were averaged over three independent replicates per strain.Animals were grown to day 1 or day 4 adult. Living animals were immobilized on a 2% agarose pad with 0.01% sodium azide, and intact neurons were scored under fluorescence microscopy on a DeltaVision Elite (GE) imaging system using an Olympus 60× oil objective. Scoring was conducted blinded to genotype. Figures show results from at least three independent replicates.C. elegans were harvested and snap frozen per sample. Protein was extracted by resuspending pellets in 1× sample buffer, three sessions of 10 s sonication with cooling on ice water in between sessions, and 10 min boiling. Samples were loaded and resolved on precast 4-15% gradient SDS-PAGE gels and transferred to PVDF membrane as recommended by the manufacturer (Bio-Rad). On immunoblots, human TDP-43 was detected by a monoclonal antibody, anti-TDP-43 . TDP-43 phosphorylated at pS409/S410 was detected by a monoclonal antibody, anti-phospho TDP-43 . Human tau was detected by a polyclonal antibody anti-tau . Phosphorylated tau was detected by a monoclonal antibody anti-phosphorylated tau . C. elegans β-tubulin levels were measured using monoclonal antibody E7  as a loading control. HRP-labeled goat anti-mouse IgG secondary antibody  was used at a dilution of 1:2500. HRP-labeled mouse anti-rabbit IgG secondary antibody  was used at a dilution of 1:10,000. Quantitation was completed by ImageJ software densitometry analysis of scanned film images.Approximately 10,000 stage-matched day 1 adult All statistical analyses were performed using GraphPad Prism statistical software. Statistical significance was determined using one-way ANOVA with Tukey's multiple-comparison test. Behavioral assays are graphed using violin plots showing the distribution and density of measures of worm movement in response to the particular stimulation for each assay; all other data are presented using standard bar graphs. Error bars represent standard error of the mean (s.e.m.).


In recent years, graphene has shown great application prospects in tunable microwave devices due to its tunable conductivity. However, the electromagnetic (EM) properties of graphene, especially the dynamic tunning characteristics, are largely dependent on experimental results, and thus are unable to be effectively predicted according to growth parameters, which causes great difficulties in the design of graphene-based tunable microwave devices. In this work, we systematically explored the impact of chemical vapor deposition (CVD) parameters on the dynamic tunning range of graphene. Firstly, through improving the existing waveguide method, the dynamic tunning range of graphene can be measured more accurately. Secondly, a direct mathematical model between growth parameters and the tunning range of graphene is established. Through this, one can easily obtain needed growth parameters for the desired tunning range of graphene. As a verification, a frequency tunable absorber prototype is designed and tested. The good agreement between simulation and experimental results shows the reliability of our mathematic model in the rapid design of graphene-based tunable microwave devices. Carbon-based materials are an important member of the material family, and their inherent excellent physical or chemical properties have attracted extensive attention in various disciplines and fields. Graphene, a planar monolayer of carbon atoms arranged in honeycomb structures, has recently sparked intense and multidisciplinary research since the advent of free-standing graphene in 2004 . In termThe early research on graphene was mostly theoretical simulation, and its EM modeling was based on Kubo’s formula . During In the microwave range, graphene can be considered a frequency-independent resistive film , and itsTo address these problems, in this work, firstly, with the help of transmission line theory, the measuring method of GSS is improved to help us obtain more precise tunning ranges of graphene. Secondly, the influence of growth parameters on the dynamic tunning property of graphene is investigated both qualitatively and quantitatively. To better reflect the numerical relation between the tunning and growth parameters, several statistical methods, including orthogonal experiment design ,30 and mCopper (25 μm) and CVD furnace (OTF 1200-X) were purchased from Hefei Kejing Material Technology Co. Ltd., Hefei, China, Hydrochloric acid  and nitric acid  were purchased from Nanjing Wanqing Co. Ltd., Nanjing, China, Hydrogen  and Methane  were purchased from Nanjing Shangyuan Co. Ltd., Nanjing, China, Polyvinyl chloride  was purchased from Shanghai Lingmin Trading Co. Ltd., Shanghai, China, Diaphragm paper (NKKTF4030) was purchased from Guangdong Canrd New Energy Technology Co. Ltd., Guangzhou, China.10H20F6N2O5S2, CAS: 464927-84-2) was purchased from Lanzhou Institute of chemical physics. Wire mesh  and Polydimethylsiloxane  are purchased from CS New Materials Co., Ltd., Jining, China.Ionic liquid (methoxyethyldiethylmethylammomium bis((trifluoromethyl)sulfonyl)-imide, C2, which can be cut into two pieces, subsequently, the two pieces can be combined into a GSS, typical photographs of synthesized graphene are shown in Large-area graphene is synthesized by CVD on copper foil. Copper foil is placed on a quartz holder in a CVD furnace. The mechanism of CVD on copper foil and the growth curve are shown in As shown in During the measurement, each GSS is measured four times for reducing test error by changing the relative position between GSS and waveguide.A is calculated throughS11 and S21 are reflection and transmission coefficients. Due to the metallic ground structure of designed absorber, the transmission is blocked. As such, S21 equals zero, so A is calculated through only S11 parameters.The absorption rate of the graphene-based absorber was tested by the waveguide method, and the waveguide used is WR62 (11.9–18 GHz). The absorption rate The dynamic tunning range of graphene is measured through the waveguide method, as shown in γ and characteristic impedance Z of the medium  in the cross-section of the waveguide can be expressed by the following Formulas (2)–(4):rε is the relative permittivity of the waveguide-filled medium, μ0 is the free space permeability, β(ω) is the phase constant, and t is the thickness of various mediums along the waveguide direction. The thickness of air part (35 mm) can be found in the datasheet of WR90 waveguide. The thicknesses of PVC and paper are 70 μm and 50 μm, respectively, and the relative permittivity of air, PVC, and paper are 1, 3, and 2.5, respectively.To accurately obtain the sheet resistance of graphene, we improved the calculation process of the previously reported waveguide method ,34 with The different transfer matrices are connected in cascade to obtain the response of the entire waveguide section as shown in Formulas (5)–(7):TTotal can be obtained from the measured scattering parameters  (°C),  (mins),  (mins),  (sccms), respectively. The measured results of the orthogonal experiment is shown in T1, Δt1, Δt2, Ratio) =  is used as the initial value of the parameter comparison experiment.To investigate the influence of each parameter, the specific parameter should fluctuate around an initial value while keeping the other three parameters unchanged (single variable principle). The first problem to be solved is the initial values of periment ,30 is usT1, Δt1, Δt2, Ratio, the second set of experiments, namely, parameter comparison, is carried out to explore the influence of a single parameter on the tunning range of graphene, as shown in T1, annealing duration Δt1, growth duration Δt2, and gas ratio are plotted in T1 will lead to relatively low growth uniformity, it also ensures the upper limit of the dynamic tunning range of graphene. For example, when the annealing temperature is 975 °C, the upper and lower limits of the dynamic range of graphene are slightly higher than those of other groups. Graphene with lower static sheet resistance can be obtained by changing annealing duration Δt1 or gas ratio, but as shown in t2, the variation trend is relatively smooth. Together with the SEM pictures in T1 or too long Δt1  can be seen as a modified model, which not only can reflect the changing trend of graphene tunning range with a single variable but also includes the effect or other three parameters, thus increasing the reliability. As shown in To effectively process the previous data and provide guidance for the subsequent work, we fitted the data and formed the mathematic model of the relationship between the dynamic tunning range and growth parameters of graphene. Different from the static sheet resistance, the dynamic range of graphene is not a single value, but an interval. To establish a quantitative model of the interval, one can choose to fit the relation among the upper, the lower bounds of the interval, and growth parameters. However, to better reflect the nature of the interval, we refer to the concept of relative bandwidth from the index of the absorber and define the concept of relative dynamic range:s domain ,40. The s domain ,32, the s domain ,42, and found in . The phyy′ represents the median of the tunning range of GSS. The values of qi,j after optimization are shown in 0 is 3.7768 × 104, and the corresponding fitting result is shown in Among them, After obtaining the mathematic models, one can not only directly predict the tunning range of graphene through the growth parameters but also obtain needed growth parameters for desired tunning range. Here, we make a simple demonstration of the second function of the model, namely to deduce the required growth parameters under the circumstance of the known desired tunning range of GSS.x1 = 1025, x3 = 20, x4 = 70. The median of the range is 0.9 kΩ/sq, substituting it to (15), x2 = 119.2, 159.3, 201.1, and 231.8 can be obtained. Except for the out-of-range value 119.2, we calculate the corresponding relative dynamic range of graphene under the other three values of annealing duration through (13). The relative ranges are 112.34%, 101.86% and 81.27%, respectively, thus the tunable range of graphene in a GSS can be obtained as  Ω/sq,  Ω/sq,  Ω/sq. We can see that the desired range is involved in the interval of  Ω/sq, namely, the tunning range of around 400 to 1400 Ω/sq can be obtained using the parameter x1 = 1025, x2 =159.3, x3 = 20, x4 = 70, which will be verified by a frequency tunable absorber shown in When considering the desired range of tunable characteristics, both (13) and (15) are needed. For instance, the desired tunning range of graphene is 400 to 1400 Ω/sq, and we predetermine that T1, Δt1, Δt2, Ratio) = , as stated in It should be noted that when using the model to predict the dynamic tunning range of GSS or deduce the needed parameters based on desired tunning range, one should still obey the rule of single-parameter principle, namely, the other three parameters should equal the optimized value obtained by the orthogonal experiment. The reason lies in that the data used to calculate to coefficients of the model, i.e., the data of the parameter comparison experiment is originated from the initial value of (p = 2.7 mm, the width of the graphene ribbon is wg = 2.2 mm, the side length of the metal ribbon is wm = 1.6 mm. The substrate adopted here is PDMS, with relative permittivity of 2.8 and the thickness of tsub = 1 mm. The thickness of the metal ribbon and bottom metal layer is tm = 125 μm, and the sheet resistance is 0.5Ω/sq. Commercial software CST 2019 was used to simulate the model, in which unit-cell boundary condition is set up. The incident wave is vertical to the sample with the electric field perpendicular to the graphene strip. In the simulation, the sheet resistances of graphene ribbons are 400, 600, 800, 1000, and 1400 Ω/sq, respectively. As can be seen from In this section, a frequency tunable absorber is designed and fabricated to verify the effect of the mathematic model on the actual device. The overall structure of the absorber is shown in x1 = 1025, x2 =159.3, x3 = 20, x4 = 70 are used to synthesize the graphene with a desired tunning range of 400 to 1400 Ω/sq. The graphene ribbon and metal ribbon are realized through a laser engraving machine, the material of the metal ribbon layer and the metal ground layer is transparent metallic wire mesh. The photograph of the metal ribbon on PDMS substrate and the patterned graphene is shown To verify the simulated results and also the correctness of the mathematical model, as calculated in the last section, the parameters In summary, the influences of CVD parameters on the dynamic tunning range of graphene were systematically investigated. The measurement method of GSS is improved to obtain more accurate tunning ranges of graphene. Direct mathematical models between growth parameters and the tunability of graphene are given. As a result, one can quickly and precisely predict the tunning range of graphene through the growth parameters or obtain needed growth parameters for the desired tunning range, which largely improves the design efficiency of graphene-based microwave devices. At the end of the work, the usability and accuracy of the proposed mathematic relation are verified by a frequency tunable absorber. The results of this work have positive significance for promoting the research and application of novel materials.


Silk fibroin and three kinds of graphene-based materials  have been widely investigated in biomedical fields. Recently, the hybrid composites of silk fibroin and graphene-based materials have attracted much attention owing to their combined advantages, i.e., presenting outstanding biocompatibility, mechanical properties, and excellent electrical conductivity. However, maintaining bio-toxicity and biodegradability at a proper level remains a challenge for other applications. This report describes the first attempt to summarize the hybrid composites’ preparation methods, properties, and applications to the best of our knowledge. We strongly believe that this review will open new doors for coming researchers. Silk fibroin (SF), a natural polymer fibrous protein extracted from silk, has been explored in many fields, such as tissue engineering, regenerative medicine, and drug delivery ,2,3,4, oGraphene and its derivatives (GDs) include few-layered graphene (FLG), ultrathin graphite, graphene oxide (GO), reduced graphene oxide (RGO), and graphene nanosheets . As a kiRecently, the combined application of SF and graphene-based materials has come to be regarded as a popular topic in biomedical fields such as tissue engineering, drug delivery, and some dental fields as shown in Graphene and graphene-based materials, as novel and promising biomaterials, have been applied in bone tissue engineering by incorporating them into various scaffolds, such as silk-based scaffolds. The combination of SF and graphene-family materials can counteract the deficiencies of each to broaden their applications in biomedical fields. SF and graphene-based derivates can be combined by either physical  binding or chemical  binding, or both . The maiFreeze-drying, a gentle dehydration method, is commonly used to dry porous scaffolds by sublimating the ice particles formed in the freezing process . The sizThe LbL assembly technique may also be employed to fabricate a kind of robust microcapsule containing silk fibroin and graphene oxide with controlled permeability . This meNanomembranes with excellent uniformity and overall integrity, containing alternating layers of SF and GO, were prepared using the spin-assisted layer-by-layer (SA-LbL) technique . To do sElectrospinning has become a standard and notable method to fabricate scaffolds recently. It has been demonstrated that scaffolds prepared by electrospinning can simulate the nanofiber bundle components of the bone ECM and wound healing ,28,29,30Vacuum-assisted assembly has been referred to as a widely used method for fabricating graphene-based flexible “paper” materials ,38,39,40A photochemical crosslinking method has been employed to fabricate tough nanocomposite hydrogel consisting of regenerated silk fibroin (RSF) and graphene oxide . The for−1, the diameter was suddenly decreased by enhanced conductivity produced in the process of electrospinning, increasing the elongational forces. Too-high graphene contents hindered the complete dispersion of graphene flakes in the solution, negatively impacting the fiber diameter nonuniformity.Parts of the areas making up the deposited graphene on the SF/GO conductive fibrous scaffolds may appear darker, indicating some small fragments of graphene being stacked . DispersTo investigate the effects of graphene on the physicochemical properties and in vivo cellular responses of silk fibroin scaffolds, Ding et al. fabricated porous 3D SF/graphene scaffolds with different contents of graphene  . It was The uniformity of topography may depend on the ratio of SF to GO. In pure configurations, GO has the appearance of a slightly cracked sheet with grooves in the configuration. Meanwhile, silk fibroin appears to have a largely smooth surface, according to previous studies . As obseThe spin-assisted layer-by-layer (SA-LbL) technique was employed and assembled a type of ultra-robust nanocomposite membrane containing GO sheets and silk fibroin . Silk fin shells, with an increasing thickness from 5 ± 0.5 nm to 23 ± 2 nm, along with an increasing number of bilayers, exhibited linear growth, indicating the absorption of the silk ionomer/GO composite only on the top of the outermost layers, and a restricted interlayer diffusion [3.5 shell permeability at different pH conditions was the key point of this research. With the increase in pH value from 2.0 to 11.5, the molecular weight of the dextrans that were permeable for the hybrid shells revealed a descending trend with the change for almost all, except 2000 to 250 kDa. In addition, the porosity of the SF/GO shells controlled by pH was much lower compared to pure silk shells. Nevertheless, previously reported robust and pH-responsive thin-shell LbL silk-based microcapsules demonstrated reversible variations in shell permeability, where the permeability was significantly enhanced in acidic and basic conditions, along with pore sizes becoming much larger, thereby accelerating the permeability of large macromolecules irrespective of environmental conditions [3.5 hybrid microcapsules can be ascribed to the transition from extended molecular chains to random coils on account of reduced charge density along with increasing pH values [3.5 composites in highly acidic (pH 2.0) and primary (pH 11.5) conditions was inconsistent with the great-volume swelling (up to 800%) of homogeneous silk ionomer capsules previously reported [The robust LbL microcapsules containing poly-amino acid-modified silk fibroin reinforced with graphene oxide controlled permeability . The plaiffusion ,58, mainiffusion . Signifinditions . The traH values ,61,62, treported . This wareported , as showThe strong SF/GO composite hydrogel films with layered structures, forming a loose three-dimensional (3D) network and mimicking the structure of nacre, relying on the force balance between electrostatic repulsion and hydrogen bonding interaction, possessed an SF uniformly coating on GO sheets, in which a single layer had lateral dimensions of several micrometers and a thickness of about 0.8 nm .Due to the GO sheets, a corrugated morphology was formed on the PLGA/TSF/GO nanofiber surfaces, resulting in micropores or mesopores . TSF plaThe porous SF/GO composite scaffolds yielded by a green fabrication method presented a continuous pore structure and a remarkable 92–94% porosity. The morphology and pore diameter partly depended on the GO content . After GIt has been demonstrated that adding graphene can enhance the mechanical features of composite materials ,67. The Among the graphene-based materials, except for the graphene, graphene oxide and reduced graphene oxide also contributed a lot to enhancing the silk-matrix biomaterials’ mechanical performance. Huang et al.  reportedAn SF-GO layered film was fabricated through another method, vacuum-assisted filtration, and showed excellent mechanical properties . A sandw−3, with the highest toughness of 3.4 MJ m−3 recorded for some samples.In contrast to the above two SF-GO composites films ,21, the n hybrid microcapsules were fabricated and exhibited remarkable stability at high concentrations of dextran solution, without noticeable buckling, over a wide range of dextran concentrations from 0 to 25%. However, the microcapsules fabricated by only silk ionomers, without graphene oxide sheets, presented partial deformation of silk-matrix capsules at the 4% dextran concentration. They even led to a collapse when the dextran concentration reached 12% [n shells. The elastic modulus of reinforced (SF-PL/GO)n shells exhibited a very high value, around 470 MPa, surpassing typical values for pure polymers in the swollen state or LbL protein shells, indicating the remarkable effect of graphene oxide on the mechanical properties of shells [Except in the form of films, the robust (SF-PL/GO)ched 12% . The cheched 12% , and a cf shells ,78.To some extent, the standard configuration of the scaffolds for tissue engineering could affect the mechanical properties of the composite biomaterials. Biomineralized poly(L-lactic-co-glycolic acid) (PLGA)/graphene oxide/tussah silk fibroin (TSF) nanofiber scaffolds with multiple orthogonal layers were fabricated through the electrospinning method . Multipl3 [4 treatment could significantly weaken the mechanical properties of the RSF/GO nanocomposites, which could probably be ascribed to the low crosslink density and the disruption of a disulfide bond in RSF.The Young’s modulus of the tough RSF/GO nanocomposite hydrogels reached as high as ∼8 MPa, and the tensile toughness was as high as ∼2.4 MJ/m3 . The RSFThe compressive moduli of a kind of SF/GO hybrid scaffold incorporated with 0.1, 0.2, 0.5, and 1.0 wt% GO fabricated via a freeze-drying green method were 19.14, 19.33, 21.73, and 24.32 kPa, respectively, higher than that of the new SF scaffold (14.02 kPa) . The uniMost SF/GO composite materials presented an improved mechanical performance compared to their counterparts. The hybrid materials were typically stiffer than pristine SF materials and tougher than GO materials . The excThermodynamic properties of G/SF conductive scaffolds were investigated by differential scanning calorimetry (DSC) to explore the effect of graphene on the crystallization of the scaffolds . ResultsElectroactive biomaterials can exert electrical stimulation on the cells growing on them. Electrochemical tests indicated that higher graphene contents endowed the G/SF scaffolds with higher conductivity and could carry electrical stimulation further to the cells .It was demonstrated that the biocompatibility of SF and GO could make them a promising biomaterial in biological scaffold and tissue engineering. The isoelectric point (pI) of SF is 3.8–3.9 , which iA hydrophilic surface of a biomaterial possesses a slight contact angle. Some studies have concluded that moderately hydrophilic surfaces (water contact angle between 20° and 55°) are beneficial to cell attachment and growth on the scaffolds ,86,87,88Water stability plays a vital role in the different applications of in vitro and in vivo scaffolds. The porous SF/GO hybrid scaffolds fabricated by a green method exhibited excellent water stability by incorporating 30 wt% and 40% glycerol into scaffolds, mainly due to the improved formation of the stable silk I and silk II crystal structures induced by glycerol, which was used as a plasticizer .p > 0.05). It can be concluded that the incorporation of graphene can transform the SF scaffolds into electronic conductors without compromising the inherent superb cytocompatibility of the SF matrix [Confocal microscopy analysis is usually adopted to reveal the morphological features of cells, and a CCK-8 assay is used to determine the viability and proliferation of cells in cytocompatibility tests. The results for SF/G composite scaffolds showed that rBMSCs displayed a spindle shape for all groups of scaffolds, meaning that the cells were widely spread on the scaffolds with distinct spread actin filaments . As for F matrix .Regarding the cell behavior of rBMSCs on the porous G/SF scaffolds , 0 and 0Graphene oxide (GO) possesses abundant functional groups and a large surface area, which can provide reactive sites for functionalization . PreviouWhen exposed to an enzyme solution, the scaffolds containing only SF or hybrid SF/GO exhibited a fracture and collapse phenomenon after 14 days of degradation and further corroded drastically at 28 days . HoweverLyu et al.  reportedSilk fibroin (SF) is a natural protein polymer and promising biomaterial. Modifications of SF have attracted growing interest in expanding SF applications. For modification of silk fibroin, some reactive reagents such as isocyanate, cyanuric chloride, and acid anhydrides have been used. Chemical modification of silk fibroin was carried out through serine amino acid residues, which showed that surface modification on native SF through serine residues was practicable and had the advantage of an increased β-sheet structure . IsocyanHowever, most modification agents have lower reactivity than isocyanate, cyanuric chloride, and anhydrides toward the reaction site of amino acid side-chains in SF protein. The most extensively utilized modification technique is the derivatization of the carboxylic acid residues via carbodiimide coupling with primary amines ,100. To A 3D printable electroconductive biocomposite bioink was fabricated based on glycidyl methacrylate silk fibroin (SB) and reduced GO, which possessed electrical conductivity, thermally stability, and the potential to become a promising biomaterial for neural tissue engineering .A conductive SF/G fibrous scaffold with 3% graphene was developed via an electrospinning technique, possessing excellent electrical conductivity and extraordinary physicochemical features, which made it a promising biomaterial for potential applications in local electric fields or local ionic currents for cell cultures, biological interfaces, or animal studies .It is widely believed that SF is a biocompatible material useful for preparing cellular scaffolds for tissue engineering. A composite of the two materials has already been proposed for several biomedical applications ,104. VarSF and graphene-family materials have been widely used in the biomedicine field of tissue repair and regeneration. The composites containing both are considered to exert better effects on tissue engineering. Although graphene-family materials have been widely introduced into the dental field, the use of the SF/graphene family can provide a more effective method . In the A compelling SF/GO film can achieve the reparation of periodontal tissues. Four dental tissues  make up the periodontium, which relies on the excellent differentiation capacity into cementoblasts, odontoblasts, and fibroblasts of periodontal ligament stem cells (PDLSCs). PDL is an essential constituent part in the attachment of teeth to the jaw, and PDL destruction could result in the loss of the tooth in severe cases of periodontitis commonly caused by a chronic inflammation . TherefoBone regeneration, rather than autologous or allogeneic bone transplantation, due to limited supply, is required in complex clinical conditions, including skeletal reconstruction of significant bone defects created by trauma, infection, or tumor resection ,107. CarA graded cGO-HA/SF biomimetic scaffold was fabricated Wang et al. by dispersing GO and hydroxyapatite with chitosan solution, blending with SF, and freeze-drying the resulting mixture . The scaAn ultrafine nanofiber scaffold was fabricated via the electrospinning method . The comNarimani et al. prepared a novel SF/GO nanocomposite scaffold through chemical crosslinking and freeze-drying . The exiAs the studies presented indicated, the synergistic effects of SF and GO on physicochemical properties and cellular behaviors provide a promising strategy in bone tissue engineering.The prepared tough photocrosslinked RSF/GO nanocomposite hydrogels, equipped with characteristics such as superb tensile mechanical properties, an increased β-sheet content, and decreased chain mobility, represent promising biomaterials. The will be usefully employed in load tissue engineering thanks to their higher tensile mechanical properties than those of certain natural elastomers  in both dehydrated and swollen states .However, a SF/GO substrate without a 3D structure may limit osteogenesis and angiogenesis compared to a 3D scaffold. Therefore, the in-vivo effects of an SF/GO composite within a 3D environment should be studied further. A novel 3D porous SF/GO nanocomposite scaffold was prepared through freeze-drying technology . Apart fMoreover, injectable SF/GO hydrogel scaffolds have been fabricated and characterized as an excellent biomedical material in bone tissue engineering . The effHowever, few studies have applied the composites of SF and graphene-family materials to cartilage tissue regeneration.Neuroregeneration is a therapeutic process of repairing the injured neurons resulting from neurodegenerative diseases. The primary condition of neural restoration is to provide nerve tissues with electrical activity, and graphene-based materials can meet this requirement . To someElectrospun SF scaffolds coated with rGO were produced as an electroactive conductor to stimulate or restore the functionality of damaged neural tissues . When cuBombyx mori pheromone binding protein (BmorPBP1) immobilizing on an rGO surface of a field-effect transistor, was developed to detect odors, among which only eugenol could elicit a strong signal [Biosensors, applied for monitoring biological processes and detecting biomolecules, are expected to meet some requirements, including having excellent physicochemical properties, biocompatibility, conductivity, and biostability . Grapheng signal .It has been concluded that the release of drugs from scaffolds is governed by several elements, such as the nature and molecular weight of the drug, degree of crosslinking density, the pore size of the matrix, solvent type, and so on . The porSalvianolic acid (SB), a traditional Chinese herbal medicine with various biological activities, has been reported to promote spinal fusion by facilitating osteogenesis and angiogenesis . An SF-Gn composite microcapsules showed excellent mechanical properties with an elastic modulus value of about 0.5 GPa, a capacity to maintain stability under extreme acid (pH 2) and primary (pH 11.5) conditions, and pH-regulated permeability [Robust (SF-PL/GO)eability , therebyeability . Beyond Silk fibroin, a natural polymer material, has been widely applied in the biomedical fields mainly owing to its advantages, such as proper biocompatibility, adaptable biodegradability, stable mechanical properties, and the ability to be modified easily. There are three types of materials in the graphene family—graphene, graphene oxide, and reduced graphene oxide—possessing excellent mechanical properties, electrical conductivity, and atomic structural stability. The combination of silk fibroin and graphene-based materials offers the potential to mix their advantages and cover each other’s shortcomings, to produce enhanced functions with more significant potential in biomedical fields. However, biotoxicity and biodegradability remain challenges that need further study. Meanwhile, there is still a need to further investigate the specific signal pathway of the mechanisms of the effects of such hybrid composites on cells. Various fabrication methods have been reported for composite biomaterials, such as freeze-drying, layer-by-layer assembly, electrochemical microstamping, crosslinking, etc. Recently, the combinations of silk fibroin and graphene family materials have shown great popularity in the biomedical fields, including tissue engineering, drug delivery, and dental fields. In oral and maxillofacial surgery, tissue engineering  has been a research topic in great demand. More efforts should be made to identify new information that supports new treatments for common oral and maxillofacial issues such as maxillofacial fractures and tumors.


The combination of graphene and TiO2 was beneficial to eliminate the recombination of photogenerated electron holes. The self-doping Ti3+ was introduced, accompanied by the crystallization of amorphous TiO2, during the hydrogenation process. Consequently, the narrowed bandgap caused by self-doping Ti3+ enhanced the visible light absorption and thus made the composites appear black. Both of them improved the photocatalytic performance of the synthesized black TiO2/graphene composites. The band structure of the composite was analyzed by valence band XPS, revealing the reason for the high visible light catalytic performance of the composite. The results proved that the black TiO2/graphene composites synthesized show attractive potential for applications in environmental and energy issues.In this study, in situ TiO This recombination of the electron hole would reduce the quantum efficiency that weakens the photocatalytic efficiency [Titanium dioxide , and various ratios of graphene were well mixed to obtain a mixture solution. The polyethylene glycol (PEG) was used as surfactant for enhancing the surface bonding between TiO2 and graphene. Then, the mixture solution was slowly dripped into a solution of 250 mL C2H5OH and 250 mL H2O while stirring. Through this process, the hydrolysis and polymerization of TBOT and H2O occurred to form a sol [Typically, the black TiOrm a sol ,20. The 2/graphene composites were obtained after drying at 80 °C for 6 h and calcining at 200 °C for 2 h in air. As shown in 2/graphene composites were calcined in H2 flow at 500 °C for 2 h under atmospheric pressure. Then the black TiO2/graphene composites  were synthesized.The above reaction was held for 1 h and then precipitated for 3 h. The precipitate was centrifuged and washed 3 times with ethanol. Subsequently, the amorphous TiO2/graphene composites were examined by high-resolution transmission electron microscope . The crystal structure of composites was detected by X-ray diffraction (XRD) on Rigaku D/MAX-2400 . In order to confirm the chemical compositions and band status of the composites, XPS spectrum was characterized on AXIS-Ultra DLD-600 W . The state of the graphene was characterized by Raman spectroscopy with LabRAM HR800  using laser excitation at 532 nm. In order to examine the light absorption range of the composites, UV–Vis absorption spectra examinations were performed on a Shimadzu UV-3600 Plus  UV-VIS-NIR Spectrophotometer.The morphology, structure and element distribution of the black TiOThe photocatalytic activity of the composites was determined in the decomposition of the methyl blue (MB). The 10 mg/L MB solution was prepared to test the visible light catalytic performance of the as-synthesized composites (BTG). A sample of 10 mg BTG was added to 100 mL MB solution and stirred in the dark for 30 min to achieve adsorption/desorption equilibrium. The visible light irradiation of the photocatalysis experiment was from a 300 W halogen tungsten lamp with a cut-off filter (λ > 420 nm). The reaction solution was controlled at 20 °C with a water-cooling system. In the photocatalysis experiment, 3 mL of reaction solution was taken every 10 min, and the catalyst was removed by centrifugation . The concentrations of residual MB were analyzed by the absorption band maximum (660 nm).2/graphene composites before and after the hydrogenation process are shown in 2/graphene composites before the hydrogenation process show only some diffuse peaks, indicating that the composites before hydrogenation are amorphous. After the hydrogenation process, the peaks occur at 25.28° (101), 37.80° (004), 48.05° (200), 53.89° (105), 55.07° (211), 62.69° (204), 68.93° (116), 70.31° (220), and 75.03° (215) (2 (JCPDS 21-1272) [The XRD patterns of TiO21-1272) ,21. The 2/graphene composites can also be characterized by Raman spectra. The Raman spectra of the BTG with various graphene contents are shown in −1 (D band), 1580 cm−1 (G band) and 2700 cm−1 (2D band) correspond to graphene [−1 (Eg(1)), 406 cm−1 (B1g(1)), 523 cm−1 (A1g + B1g(2)), and 646 cm−1 (Eg(2)), which matched with the characteristic peaks of anatase TiO2 [2, the Eg(1) mode shifted from 144 cm−1 of bare bulk TiO2 to 156 cm−1 of BTG. The shift toward high frequency indicated the ultra-dispersed characteristics of the TiO2 nanoparticles and their combination with graphene, and the disappearance of the graphene 2D band in the BTG may be attributed to the composite of graphene and TiO2. From the Raman analysis, the characteristic peaks of TiO2 and graphene appeared in the spectra of BTG, indicating that the black TiO2/graphene composites were successfully synthesized.The structure of black TiOgraphene . For all2/graphene composites are shown in 3/2 and Ti 2p1/2 of TiO2 were revealed at 457.8 and 463.3 eV. The Ti 2p3/2 peak of the BTG shifted from 458.6 eV to a lower binding energy corresponding to the presence of a high Ti3+ concentration [2 was not doped with carbon. The curve fit of C 1s spectra of BTG is shown in 2 is a well-known catalyst.The XPS spectra of the black TiOntration ,25,26,272 in the composites was anatase structure. The SAED result was consistent with the XRD characterization, which indicated that the TiO2 in the composite was anatase structure with excellent photocatalytic activity. 2/graphene composites. It can be seen that the size of individual TiO2 nanocrystals was approximately 15 nm in diameter. There was a disordered surface layer surrounding the TiO2 nanocrystal, as shown by the dotted red circle in 2 reported by Chen et al. [2 nanocrystal was created by hydrogenation, which caused a significant color change and enhancement of visible light photocatalytic activity. The schematic diagram of the sample color change (from blue to black) after hydrogenation is shown in 2. The energy dispersive X-ray (EDX) elemental mappings of Ti, C, O taken from the STEM image of 2 on graphene in the black TiO2/graphene composites.The selected area electron diffraction (SAED) pattern is shown in the inset of n et al. . The dis2/graphene composites. The presence of graphene significantly improved the visible light absorption of the black TiO2/graphene composites. The visible light absorption intensity of the composites was enhanced with increasing graphene content. In order to characterize the band gaps of the composites, the Kubelka–Munk function (F(R∞)·E)1/n versus the energy of light (E = hv) is shown in 2, n = 2 will give the best linear fit. As the graphene content increased from 1% to 15%, the band gaps were estimated roughly to decrease from 3.05 to 2.94 eV. It is well known that the band gap energy of anatase TiO2 is 3.2 eV. The band gap around 3 eV of the composites was lower than that of anatase TiO2, which was attributed to the self-doping of Ti3+. Moreover, the composites had enhanced light absorption in the range of visible light, which was consistent with the darker sample color with increasing graphene content. The results suggest that both graphene combination and self-doping of Ti3+ play a crucial role in the photocatalytic activity of the composites. 0 and c are the MB concentration before and after visible light irradiation, respectively. The k value in the formula represents the photocatalytic reaction rate. Through fitting calculation, the photocatalytic reaction rates k for BTG-1, BTG-5, BTG-10 and BTG-15 were determined to be 2.88, 3.61, 3.99 and 3.23 h−1, respectively. The BTG-10 exhibited the highest photocatalytic activity. To determine the recyclability of the composites, the BTG-10 was recycled under several visible light irradiation cycles. As shown in 2/graphene composites, BTG-10 was selected for valence band (VB) XPS analysis in 2 (3.0 eV). The insets in 2 nanocrystal introduced by hydrogenation can upshift both the VB and CB edge of the TiO2/graphene composites. According to the energy band analyses, the VB of the BTG-10 was higher than that of the O2/H2O, and the CB was lower than H+/H2, potentially suggesting that the composites have attractive potential for applications in environmental and energy issues.Since BTG-10 had the best catalytic activity of the TiO2/graphene composites is shown in 2 under visible light irradiation. It is well known that the graphene to which the nano-sized black TiO2 attached has good electrical conductivity [2. This hinder effect is the reason why BTG-10 had a higher catalytic activity than BTG-15.The photocatalytic mechanism of the black TiOuctivity . Therefo2/graphene composites with different graphene contents were successfully synthesized. In the sol–gel process, TiO2 was generated in situ on the surface of graphene from TBOT as a titanium source. The good conductivity of graphene is beneficial for eliminating the opportunity for photogenerated electron-hole recombination. In the hydrogenation process, self-doping Ti3+ was introduced, accompanying the crystallization of amorphous TiO2. The narrowed bandgap (2.94~3.05 eV) caused by self-doping Ti3+ enhanced the visible light absorption. Moreover, the nanostructured black TiO2-graphene composites showed enhanced visible light photocatalytic activity in methyl blue degradation. The sample with 10 wt% graphene showed the highest photocatalytic activity and good stability. The incorporation of black TiO2 caused by hydrogenation and graphene composite expanded the light absorption range and reduced the recombination of photogenerated electron holes, both of which enhanced the capacity of photodegrading organic dyes. Therefore, this work is expected to open up a new way for the synthesis of black TiO2/graphene composites, and its high photocatalytic activity proves that the black TiO2/graphene composites have attractive potential for applications in environmental and energy issues.A series of black TiO


Advanced nanoelectromechanical systems made from polymerdielectricsdeposited onto 2D-nanomaterials such as graphene are increasinglypopular as pressure and touch sensors, resonant sensors, and capacitivemicromachined ultrasound transducers (CMUTs). However, durabilityand accuracy of layered nanocomposites depend on the mechanical stabilityof the interface between polymer and graphene layers. Here we usedmolecular dynamics computer simulations to investigate the interfacebetween a sheet of graphene and a layer of parylene-C thermoplasticpolymer during large numbers of high-frequency (MHz) cycles of bendingrelevant to the operating regime. We find that important interfacialsliding occurs almost immediately in usage conditions, resulting inmore than 2% expansion of the membrane, a detrimental mechanism whichrequires repeated calibration to maintain CMUTs accuracy. This irreversiblemechanism is caused by relaxation of residual internal stresses inthe nanocomposite bilayer, leading to the emergence of self-equilibratedtension in the polymer and compression in the graphene. It arisesas a result of deposition–polymerization processing conditions.Our findings demonstrate the need for particular care to be exercisedin overcoming initial expansion. The selection of appropriate materialschemistry including low electrostatic interactions will also be keyto their successful application as durable and reliable devices. A wide range of graphene-based NEMS devices havebeen demonstrated,2 including capacitivepressure sensors, microphones, capacitive micromachined ultrasoundtransducers (CMUTs), and accelerometers. Nevertheless there are somelimitations encountered with graphene such as the low device yielddue to damage or the contamination of graphene during chemical vapordeposition growth and transfer from the parent to the target substrate.3 Recently, this has been overcome by employinga laminated hybrid membrane comprised of graphene and an ultrathinpolymer layer, resulting in 100% yield of high performance grapheneNEMS pressure sensors4 and acoustic transducers.5 Ultrathin bilayer nanocomposites are thereforepromising materials also for higher frequency devices such as CMUTsand resonant sensors. One of the polymers of choice, parylene-C, hasalso been demonstrated as an effective dielectric for graphene nanoelectronicdevices such as field effect transistors6 and memristors.7 However, the mechanicalstability of the combination of graphene and parylene-C  owing to its combinationof superlative properties,ne-C see 1a,b as uFor application of nanocomposite laminates as CMUTs,and more generallyas NEMS, the mechanical properties of the biphasic nanomaterial mustremain stable, that is, unchanged for a given loading range. In thecase of mechanical properties, the material must remain elastic. Inelasticityof the nanocomposite mechanics causes sensor measurements drift andresolution issues and opens questions as to how often resulting additionalcalibration steps are required. One of the main causes of mechanicalirreversibility is the degradation of the interface between polymerand graphene layers. Of course, the polymer itself is likely to dissipatemechanical energy under external loading; however, the interface constitutesthe most likely location for slippage or void growth to occur. Themechanics of the polymer–graphene interface under externalloading must therefore be investigated to ensure long-lasting calibrationand durable application of layered nanocomposites.3 during the polymerization–deposition fabricationprocess and during operating conditions consisting of the applicationof transverse acoustic waves  simulationsparticularly suited. MD simulations provide access to local and high-resolutionmechanical data at the interface typically inaccessible via characterizationexperiments only. We therefore make use of MD simulations to focuson the dynamics of a bilayer nanocomposite volume of approximately1000 nmFigure S1), graphene,12 and graphene–polymernanocomposite9 mechanical properties. Moreover,their statistical robustness is ensured by the use of ensembles constrainingthe intrinsic aleatoric uncertainty of MD.14Theaccuracy of the prediction of our MD simulations is underpinnedby their ability to reproduce parylene-C  interactionsas well as pairwise  interactions. TheMD simulations were performed using the widely used LAMMPS code.18Classical molecular dynamics (MD)simulations were performed usingthe optimized potentials for liquid simulations (OPLS) force field,https://github.com/velocirobbie/make-graphitics/).19MD simulationsof deposition, polymerization, densification, equilibration, and loadingare performed for several polymer nanocomposite formulations. Thereference membrane in our study is assembled from a layer of parylenetype C monomers deposited and polymerized onto an infinite graphenesheet see 1a,b. ParCMUT usage involves the application of orthogonal pressure to themean plane of the bilayer nanocomposite membrane see 1c. OrthoSensing high-frequency pressure waves of MHz range means that themembranes face cycles of loading and unloading completed in the rangeof 10 ns or 1 μs. Further, as we want to observe the stabilityof the interface between the two constituents of the bilayer, we needto simulate enough cycles of loading for the irreversible mechanismsto occur. We simulated up to 50 loading cycles. Inherently, withoutany additional assumption, each individual replica MD simulation isused to compute the dynamics of the membranes for at least 100 nsto 1 ms.20 We verifiedthe convergence of the mechanical response of the material with decreasingstrain rates. We actually determined that exactly the same variationsof the three dimensions were found when applying in-plane stress tothe membrane, whether it is applied over 10 ns or at least 100 ps. Below 100 ps per cycle,i.e., at very high strain rates, elastic strains are not able to propagatein the system, overestimating its stiffness; we therefore observeunderestimated strains during cycles of pressure controlled stretch.Based upon these observations, we assume that we can speed up ourMD simulations 50-fold. Each cycle of loading and unloading is thereforeapplied in 200 ps instead of 10 ns.However, we checked for potential separation of timescales inour molecular models of the membrane, which is often observed forthermoplastic polymer systems.x and y dimensions. In the vertical dimension z, the systemconsists of one sheet of graphene superimposed with an approximately10 nm thick layer of polymerized parylene-C (after compaction). Thetwo contacting layers are surrounded by a 5 nm thick void .21 The graphene sheet is inserted horizontallyin a periodic box filled with monomers, parallel to the x–y plane. The resulting systems contain 5000monomers of parylene, each containing 16 atoms and one graphene sheetof 3680 atoms, for a total of 83 680 atoms.In more detail the first step consists of randomlypacking monomersof parylene within a large 15 × 15 × 15 nmNVT), MD simulationat 600 K. For every 250 bonds formed, a longer relaxation of the systemis simulated, at constant temperature and pressure (NPT), for 10 ps at 1 bar and 300 K. Polymerization is terminated whenno more pairs of terminal carbons are found (<6 Å) and lessthan 1% of terminal carbons is left unbonded. At this stage, the additionalelectrostatic charges on terminal carbons are removed. The polymerizationworkflow is also facilitated by the use of the Polymatic software.During all our MD simulations, the time step is 1 fs and the dampingparameter for temperature and pressure is 100 fs.Polymerizationis performed in the second step by covalent bondingof two terminal alkene carbons of different molecules, when foundat a distance below 6 Å. The two candidate terminal carbons ofall the monomers are initially enriched with a positive or negativeelectrostatic charge, respectively, to accelerate aggregation of pairsof candidate atoms. Polymerization is performed one bond at a time,for the closest pair of alkene carbons. Additional electrostatic chargesare removed for newly bound atom pairs. Every 50 bonds formed, thesystem is briefly equilibrated and relaxed by means of a 1 ps long,constant volume and temperature . Both thethird and fourth steps are performed using NVT MDsimulations of 100 ps each.The nanocompositesystem is then compressed vertically in the z dimension. Finally, the nanocomposite membrane is equilibratedfor 2.5 ns by means of a constant pressure and temperature MD simulation,at 1 bar and 300 K, to enable the relaxation of newly formed polymerchains. The membrane is then assumed to be at equilibrium since thepotential energy of graphene and parylene-C and the dimensions ofthe system do not vary significantly anymore .An additional 5.5 nm void is insertedartificially between thebottom face of the graphene sheet and the top of the parylene layer,during the fifth step. This is done so as to avoid self-interactionof the nanocomposite through the periodic boundary conditions in the 20With this deposition–polymerizationprocess, we generateone replica of our nanocomposite molecular model. Different replicasare generated by the same process and differ only by the choice ofthe random seed. In turn, for each nanocomposite chemical formulation,we simulate ensembles of replicas of our molecular model in orderto produce robust statistics. We determined in earlier studies ofmaterial properties predictions using MD simulations that with highlyhomogeneous systems such as polymers, aleatoric uncertainty can becontained with only a small number of replicas, as low as 5.14 We compute the evolution of the in-plane dimensions Lx and Ly of the membrane throughout the cycles of loadingin response to the applied negative pressure  alsoincrease. We quantify the accumulation of permanent axial strainsϵxxp and ϵyyp in both in-plane directions, which are the remaining strains atzero-loading . We infer that permanent strainscannot be associated with further relaxation of the system. The manifestationof permanent strains in the unloaded bilayer membrane reveals an underlyingdissipative mechanism, which at this stage needs to be clarified.The dissipative mechanism could well be associated with parylene itself or the interface between parylene and graphene.We consider the relevant loading range of CMUTs to be transversepressure-controlled cycles of loading associated with acoustic wavesimpacting the membrane  and after the completeequilibration of the processed membrane (step 5) and before any cyclicloading (see We compute the average in-placestresses σding see 3d. In thxx or yy) and 3D (bulk and shear) elastic moduli ofthe bilayer with the number of cycles of loading. Reduction in internalstresses causes parylene and graphene to be in the same mechanicalstate, that is, simultaneously in tension or in compression. In turn,both materials tend to oppose one another more synchronously, enhancingthe mechanical properties. Elastic mechanical properties are directlylinked to the bending stiffness of the pressure sensor; thereforethis needs to be taken into account during the calibration of thesensing device.The decrease of potential energy in the parylene seen after multiplecycles of loading is associated with the release of the initial positiveresidual stresses within the polymer. This finding supports the emergenceof a slippage mechanism at the interface between graphene and paryleneinduced by cycles of loading. The slippage mechanism is found to appearearly on after the first high-frequency acoustic waves effect arefelt on the bilayer membrane. The mechanism continuously alters theeffective mechanical properties of the nanocomposite during firstuse cycles see 4. We obsThe hypothesis of self-equilibrated residual stresses in the membraneconstituents during processing by deposition–polymerizationis further tested by removal of one of the two constituents see 5a. When 3, and for each cellwe compute the local density of parylene. We study the distributionof these local densities in individual planes at constant distancefrom the graphene sheet. We find that the distributions tend to shrinkas we move away from the sheet. That is, the local densities are moreheterogeneous near the graphene and in particular below a 3 nm distance,which further supports the hypothesized pinning mechanism. The distributionsappear to have converged beyond 4 nm.In order to further test the pinning mechanismhypothesis, we lookfor anomalous polymer behavior near the interface with graphene. Inparticular, we observe the distribution of polymer local density nearthe interface see 5b, data We now modify the processingof the bilayer membrane to inspectthe effect of contact surface area between parylene and graphene.More precisely, we build new bilayer membranes, but this time step3 is removed as we do not compress the polymer onto graphene betweenpolymerization and equilibration. We compare the interface energybetween the two constituents, in the normal (compacted) and the modified(loose) processing conditions see 5c. We fiInstead of removing the compaction stepduring the processing,we now modify the chemistry of the polymer. We employ a more polarizedpolymer, that is, parylene-F see 1a with fWe have investigated the durability andmechanical stability ofbilayer membrane nanocomposites comprised of parylene-C and graphenewhen they are employed as sensors and transducers by simulating processingand operating conditions using classical molecular dynamics. We foundthat the mechanical properties of the nanocomposite evolve rapidlyduring operation when subjected to stretching cycles correspondingto the effect of sensed acoustic waves. By unraveling the underlyingmechanism, we proposed a solution to achieve mechanical stability.In particular, the nanocomposite accumulates a permanent strain rapidlyafter the onset of external loading. We discovered that the permanentstrain is the consequence of slippage at the interface between themembrane constituents. We found that slippage is due to the releaseof elastic energy stored as residual interfacial stresses in the polymerand the graphene layers, akin to a pinning mechanism that arises duringthe deposition–polymerization process. Due to this pinningmechanism, graphene maintains the polymer in an out-of-equilibriumstate, resulting in non-negligible internal tensile stresses. We identifiedthe dependence of these stresses upon adhesion and electrostatic forcesacting between the parylene-C matrix and graphene. The pinning mechanism,the associated interfacial stresses, and their subsequent relaxationupon operation are detrimental to the durability and accuracy of capacitivemicromachined ultrasound transducers and to nanoelectromechanicalsystems in general. To mitigate these issues, we recommend the useof polymer dielectrics displaying low electrostatic interactions withgraphene to relieve the interfacial stresses and ensure mechanicalstability. Our findings demonstrate the need to carefully select thechemical composition of these membranes for the future design of high-accuracyand reliable pressure-sensing devices based on ultrathin layered polymer–graphenenanocomposites.


Primary care providers face challenges in recognizing and controlling hypertension in patients with chronic kidney disease (CKD). Clinical decision support (CDS) has the potential to aid clinicians in identifying patients who could benefit from medication changes. This study designed an alert to control hypertension in CKD patients using an iterative human-centered design process.In this study, we present a human-centered design process employing multiple methods for gathering user requirements and feedback on design and usability. Initially, we conducted contextual inquiry sessions to gather user requirements for the CDS. This was followed by group design sessions and one-on-one formative think-aloud sessions to validate requirements, obtain feedback on the design and layout, uncover usability issues, and validate changes.This study included 20 participants. The contextual inquiry produced 10 user requirements which influenced the initial alert design. The group design sessions revealed issues related to several themes, including recommendations and clinical content that did not match providers' expectations and extraneous information on the alerts that did not provide value. Findings from the individual think-aloud sessions revealed that participants disagreed with some recommended clinical actions, requested additional information, and had concerns about the placement in their workflow. Following each step, iterative changes were made to the alert content and design.This study showed that participation from users throughout the design process can lead to a better understanding of user requirements and optimal design, even within the constraints of an EHR alerting system. While raising awareness of design needs, it also revealed concerns related to workflow, understandability, and relevance.The human-centered design framework using multiple methods for CDS development informed the creation of an alert to assist in the treatment and recognition of hypertension in patients with CKD.The online version contains supplementary material available at 10.1186/s12911-022-01962-y. Among patients with CKD, 52% have diagnosed hypertension, 19% have pre-hypertension, and 16% have undiagnosed hypertension [Clinical decision support (CDS) provides “clinicians or patients with clinical knowledge and patient-related information, intelligently filtered or presented at appropriate times, to enhance patient care.”  While raPrevious goals of CDS design have centered around efficiency, error rates, and guideline adherence rather than the overall usability of CDS features , 22. WhiIn this study, we followed a human-centered design (HCD) process that solicits feedback from clinicians at multiple stages and using multiple methods. Human-centered design can be defined as “an approach to interactive systems that aims to make systems usable and useful by focusing on the users, their needs and requirements, and by applying human factors/ergonomics, and usability knowledge and techniques. This approach enhances effectiveness and efficiency, improves human well-being, user satisfaction, accessibility and sustainability; and counteracts possible adverse effects of use on human health, safety and performance.”  Human-ceThe use of CDS has promise in the context of CKD, with recent studies showing that it leads to an increase in the rate of urine albumin monitoring , 40. HowTo design and develop the alerts, we followed a human-centered design process focusing on the involvement of users at each stage of design and development, an understanding of user needs and an iterative process Fig. . Prior tPrimary Care Physicians (PCPs) from Brigham and Women’s 15 affiliated primary care clinics were emailed a recruitment letter and frequently asked questions document by the principal investigator. Based on the simplicity of the system interface and the iterative process, the recruitment goal was a minimum of 18 total participants with 6–8 per activity until we reached saturation in our findings , 42. A rBrigham and Women’s affiliated primary care clinics use Epic Hyperspace  as the EHR. Best Practice Advisories (BPAs) were used as the format of CDS for alerting providers and giving treatment recommendations. Our decision support was intended to address three categories of antihypertensive treatment in CKD: initiation of ACE/ARB treatment, increasing dose, and adding a diuretic –46. ProvWe gathered user requirements through contextual inquiry sessions. The goal of the inquiry sessions was to understand the different activities, steps, and thinking processes involved in managing uncontrolled blood pressure using the EHR, to generate user requirements for CDS.The contextual inquiry sessions were conducted at the participant’s office or virtually, using their own computer with the software that they use to do their daily work. The moderator provided the participant with a short introduction that included information about the structure of the session and demographic questions. Participants were interviewed on their use of the EHR to manage chronic disease, the overall structure of their visits, and any challenges or issues important to them in regard to hypertension. During the second part of the session, participants were asked to show and explain their process of how they prepare for and then go through an encounter with a patient with CKD and uncontrolled hypertension, their workflow and how they use the EHR to support their activities. Clinical scenarios were used to understand how the providers interact with the EHR to support their decision making related to the patient’s management of CKD and hypertension.Based on the user requirements elucidated in the contextual inquiry sessions, we designed several mockups of the alerts in categories 1 and 2 . Not all user requirements could be incorporated due to limitations of the EHR. The structure of each mock-up included the rationale and relevant statistics, guidelines, order options and acknowledge reasons  in patients already prescribed the maximum dose of an ACE/ARB). We developed working versions of the alerts that would trigger in the sandbox environment of the EHR to use during the next phase of usability testing.We conducted individual formative think aloud usability testing sessions to uncover additional usability issues and validate design decisions. We developed five scenarios in which one of the five alert variations would trigger  reviewed the recordings of the contextual inquiry sessions to identify common themes across participants. The themes were reviewed with the broader research team. Themes were then translated into user requirements. Potential solutions to address these requirements in the CDS were brainstormed and subsequently investigated for feasibility within the EHR by the research team.Group design sessions were transcribed. Transcriptions were reviewed by two members of the research team  who independently coded participant comments by feature/functionality and design element and came together with the human factors specialist to review discrepancies, revise codes, and reach consensus. The human factors specialist (PG) reviewed the coded comments and grouped them into 7 major themes.Two research team members  reviewed the transcripts and video of the usability tests to identify usability issues encountered by the participants during the testing sessions. The research team members documented the individual user feedback, an associated quote, and contextual information describing the usability issue . A conteThroughout the design process, we held standing meetings with the research team and 1–2 additional subject matter experts in nephrology and informatics. Subject matter expert meetings were used to review user feedback, discuss requirements and make preliminary decisions about content and design. Build meetings were used to discuss the limitations of Epic and technical issues. All Team meetings included multiple highly experienced informatics researchers and clinical trial experts and was the final decision-making body.Recruitment emails were sent to 212 PCPs. Of the respondents that indicated interest in participating in the human-centered design process, 6 took part in the contextual inquiry session, 9 in the group design sessions, and 8 in the individual think aloud sessions. 3 of the contextual inquiry participants also partook in the individual think aloud sessions for a total of 20 participants. 8 of the 20 participants were female and 12 were male. All were PCPs consisting of 18 physicians, 1 nurse practitioner and 1 physician assistant  incorporating the CDS with both the clinical workflow  and clinical decision-making process, (2) providing actionable and clear recommendations, (3) including relevant contextual information, and (4) providing a simple and efficient interface. Our study showed that participation from users throughout the design process results in feedback that can be translated into user requirements and validation for design decisions.Challenges related to the adoption of CDS such as alert fatigue and provider burden are well known. Our findings, along with those identified in other studies, highlight improvements to CDS that include removing extra clicks, allowing deferral to a later date, adding more labs and their specific dates, and making the alerts more concise and visually appealing , 42. MorIn addition, some of the decisions we made addressed clinical areas without official contraindications but are common concerns. We added disclaimers about the acceptability of an increase in serum creatinine after starting certain recommended medications and a warning about teratogenicity risk for women of childbearing age.In some cases, the provider preferences we discovered differed from previous research. For example, we previously learned that PCPs wanted a reminder to think about a referral or e-consult to nephrology and this was confirmed by primary care stakeholders during our design process . HoweverWe were not able to fully address some of the more frequently identified issues due to constraints of the EHR system. Ideally, we would be able to create a system that met all the user’s requirements. Providers have diverse encounter workflows. The alert firing at the start of an encounter may be a significant roadblock for some providers and lead them to ignore the alert . HoweverWhile the human-centered design process outlined in this study can apply to a wide range of settings, there may be some limitations to the final alert design due to the context of testing. Based on local prescription trends, we included a diuretic rather than a calcium channel blocker as the next line agent and we chose hydrochlorothiazide over chlorthalidone. These trends may vary by region or provider. The testing process was limited due to the COVID-19 outbreak, causing all sessions to be conducted remotely. As a result, we were unable to perform near-live simulation and the circumstance of being behind schedule in clinic, which impacted in-person user requirements gathering and could impact the interaction with the alert. Provider understanding of a real patient’s history may also impact their treatment plan. In addition, we were limited by our own institutions governance structure in terms of what technology solutions were allowed. Finally, the providers that agreed to participate in this testing could represent a group that would be more willing to spend the time to read through the information included in the alerts.By involving the user at multiple stages of design, we were able to refine our alerts to make them better suited for clinical decision making by providing relevant data, justification, treatment options, and acknowledge reasons. While we used this HCD framework to design CDS tailored to PCPs using Epic in the context of CKD recognition and treatment, this method can be an effective way to discover user needs for CDS regardless of the EHR or clinical scenario.Additional file 1. Appendix A Usability Test Script.