Graphene oxide/rubber composites were experimentally investigated for obtaining their thermal properties. Three kinds of the composite matrix material have been used: NBR, HNBR and FKM. The reduced graphene oxide in the form of crumped flakes has been applied as the filler influencing on thermal conductivity of the composites. Two values of graphene oxide weight concentration have been taken into account in the investigation. Thermal conductivity of the composites and basic matrix has been measured by the professional apparatus with the use of the guarded heat plate method. Before measurements the preliminary tests using the simplified comparative method have been performed. The results obtained, both from preliminary tests and using the guarded heat plate method, show an increase in thermal conductivity with increasing the reduced graphene oxide content in the composite. The experimental investigation allowed to determine not only the increase in thermal properties of graphene oxide/rubber composites compared to the basic matrix, but also the absolute values of thermal conductivities. Additionally, the SEM analysis showed that the tested composite samples contain agglomerates of the rGO nanoparticles. The occurrence of agglomerates could affect the composite thermal properties. This was noticed in the comparatively measurements of the temperature of different composites during the heating of samples tested. The maximum enhancement of thermal conductivity obtained was about 11% compared to the basis matrix of the composites tested. Thermal properties of the composites considered have been studied among others in7 where the authors reported the results of thermal conductivity investigations for the graphene oxide/rubber composites. The authors in2 present the results of measurements of thermal conductivity of bromobutyl rubber composites with the ionic liquid modified graphene oxide. Weight concentrations of the graphene oxide in the composites tested were: 1; 2; 3 and 4%. The authors reported a maximum one, threefold improvement in thermal conductivity with 4% content of graphene oxide, compared to the basic matrix. The dependence of the composites thermal conductivity on graphene oxide concentration was approximately linear. In turn the study3 includes the results of thermal conductivity measurements in the case of graphene oxide/rubber composite with the basic matrix of carboxylated acrylonitrile butadiene rubber. The authors investigated the graphene oxides with different oxidation degrees. The graphene oxide content in the composites considered was 4 phr (parts per hundred rubber). The authors reported the increase in thermal conductivity of the composite in the range from 10.1 to 27.2% depending on the graphene oxide kind. The article4 deals to the graphene oxide/natural rubber composite which was prepared by a novel in-situ method. The authors reported 36% increasing in thermal conductivity of the composite compared to the pure rubber. Despite the low content of the reduced graphene oxide (0.1 phr) the high enhancement of thermal properties has been observed. In turn the work7 deals to investigations of the interfacial thermal transport in graphene oxide/butadiene-styrene-vinyl pyridine rubber composites by molecular dynamics simulation. The authors concluded on the best oxidation degree of graphene oxide about 15%. For this value the highest thermal conductivity of the composites is achieved.The rubber is used extensively in many engineering applications. To improve the material properties rubber-based composites are formed. Both the natural rubber and the synthetic rubber copolymers are used as a basic matrix in composite materials. One of the rubber applications is the use as a material of the roller bearing seals. Nanoplatelets derived from different forms of carbon have recently attracted attention as an useful filler in rubber composites. The filling of the composite with nanoplatelets improves the mechanical properties of the roller bearing seals. Additionally thermal properties are also enhancement which contributes more intensive heat dissipation generated in the bearing. The different form of carbon are used as the filler in rubber-based composites. There are: graphene oxide13 included investigations in thermal conductivity of graphene/rubber composites or graphite and carbon black as fillers. In all cases considered the increase of thermal conductivity has been observed.The another above quoted articlesThe main goal of the present work was to determine the thermal conductivity of the selected reduced graphene oxide/rubber composites applied as a material on roller bearing seals. Because of practical applications of the materials considered the knowledge of their thermal properties is important and useful. Enhancement in thermal conductivity by adding the filler in the form of the reduce graphene oxide improves the heat rejection from the bearings. Matrix materials of tested composites were acrylonitrile butadiene rubber (NBR), hydrogenated acrylonitrile butadiene rubber (HNBR) and fluoroelastomer (FKM). Due to the heterogeneity of materials investigated, special attention was paid to the measurement technique. Thermal conductivity of the composites and the basic matrix has been tested with the use of the guarded heat plate method. Discussion on the measurements techniques for thermal conductivity of not homogeneous materials confirmed the validity of the method applied.3, the specific surface area of 266 m2/g, the specific electrical conductivity − 24 S/cm2. The scanning electron microscope (SEM) images of the graphene oxide nanoparticles are presented in Fig. The reduced graphene oxide (rGO) used as the filler influencing on thermal conductivity of the composites investigated has been purchased from Institute of Electronic Materials Technology, Warsaw, Poland. The considered graphene oxide has been reduced with sodium hypophosphite. The basic properties of the rGO included: the appearance as the grey to black powder, hydrophobic and stable in air, the bulk density equal to 0.019 g/cm2 carbon of C=C bonding11. Other peaks are much smaller what indicates the considerable deoxygenation by the reduction process of the graphene oxide1. The XPS spectra obtained is comparable with the another types of rGO also applied as thermal conductive filler in rubber graphene composites4.As can be seen the rGO material consists of thin, crumpled flakes with diameter of a few micrometers which forming the loose, disordered, crumbling structures. The X-ray photoelectron spectroscopy (XPS) of the rGO considered is shown in Fig. 15 depending on the form of GO. In the case of the rGO used in the investigations the value of thermal conductivity has been estimated on the basis of the model of the effective thermal conductivity equation for two-component material. The results obtained have been included in Chapter 3—Results and discussion.The reduced graphene oxide applied as the filler in the rubber composites has a high thermal conductivity. The literature data gives values of GO thermal conductivities from several to over two thousands W/(mK)mBM is a mass of basic matrix.As the basic matrix of composites three types of rubber have been used. There were: acrylonitrile butadiene rubber (NBR), hydrogenated acrylonitrile butadiene rubber (HNBR) and fluoroelastomer (FKM). For the fabrication of rGO/rubber composites the conventional mechanical method enabling the relatively uniform dispersion of the rGO flakes in the rubber matrix has been used. The suitable amount of rGO in the form of crumpled flakes were directly dispersed to the basis rubber during the rolling process with the use of two-roll mill calendaring. The process has been performed on the laboratory roll-mill of 400 mm width and 150 mm diameter cylinder at temperature 70 °C for about 10 min. The two-roll mill applied has provided the most homogenously disperse the reduced graphene oxide in the rubber examined. After the rolling process the weight of the composite formed has been determined and the weight concentration of rGO has been obtained from the equation3. Before thermal investigations the experimental research on some mechanical properties of the composites considered have been performed. As the results the values of indentation hardness and elastic modulus have been received. Mechanical properties were investigated with the use of Ultra Nano Indentation Tester. The basic characteristics of the composites tested are included in Table The density of the graphene oxide/rubber composites have been determined using the volumetric weighing method. An accuracy of density measurements was estimated at ± 0.003 g/cmA chemical characterisation of the composites have been performed using the energy-dispersive X-ray spectroscopy (EDS). The samples for EDS analysis have been prepared directly from the rGO/rubber composites. Before investigations the external surfaces of samples tested were cleaned and degreased in ultrasonic bath of ethanol. Figure As can be seen in Fig. The next test enabling the characterization of rGO/rubber composites was the scanning electron microscope analysis of the cross section of the materials tested. Before investigations the samples were cleaned and degreased in ultrasonic bath of ethanol. Figures Scanning electron microscope observations of the filler dispersion and fracture surfaces have indicated that in the case of NBR and HNBR composites the relatively uniform distribution of reduced graphene oxide flakes occurs. However, there are agglomerates and their clusters. In the case of HNBR matrix material, where the hydrogenation process increases temperature resistance, the rGO flakes are less visible than for NBR. This makes it difficult to analyze the distribution of nanoflakes.In turn, in rGO/FKM rubber composites, the rGO flakes are even less visible, especially at low magnifications. The compaction of the flakes at the same rGO concentration is much smaller than in the case of NBR composites. This may indicate the presence of a large number of agglomerates of significant size.Measurements of thermal conductivity of the rGO/rubber composites with different graphene oxide contents were carried out in two stages. In the first stage, as the preliminary investigation, the comparative method has been performed. The obtained results encouraged further research. The second stage concerned the main measurements using the professional procedure.The preliminary investigations of thermal properties of the rGO/rubber composites had a qualitative character. They were performed on the test stand which has been constructed especially for this purpose. A view of the stand applied is shown in Fig. d = 29 mm and thickness δ = 13 mm. Such dimensions gave a relatively large sample volume which ensured that it can be assumed the same thermal properties in all sample volume, even if the distribution of reduced graphene oxide flakes is not uniform. Temperature detectors have been placed at the depth of 5 mm. The first stage of the experiment was to investigation the changes in time of the sample temperature during the sample heating. Measurements have been performed for the rubber being the basic matrix and for the rGO/rubber composites with different graphene oxide content. The comparative analysis showed the filler influence on thermal conductivity of the composites.The tested samples had a circular shape with diameter The second stage of the experiment was to determining the thermal conductivity of samples tested with the use of standard method on the professional apparatus.16, transient plane source method8 or transient hot wire method17. The alternative method applied in composite thermal conductivity investigations is a molecular dynamics simulation7. The laser flash method enables thermal diffusivity obtaining, therefore, for determine the thermal conductivity the specific heat and density measurements are necessary. The small size of a test sample is an additionally challenge both in diffusivity and heat capacity measurements. In the case of laser flash method the sample dimensions are about δ ≈ 1 mm, d ≈ 12 mm. In turn, measurements of the specific heat with the use of differential scanning calorimeter require samples of about 10 mg weight. In this case, if the material tested is heterogeneous, test results could be unreliable. In turn, the transient methods, both the plane source and the hot wire, require the infinite in all direction and isotropic test sample20. Ensuring these conditions is very difficult. The authors in4 applied the plate-shaped samples of rGO/natural rubber composite with side of 254 mm and thickness of 50 μm. Such dimensions approximate infinity in only two directions. The very small thickness of the sample could significantly affect the measurement results. The second transient method—the hot wire—requires placing the probe being the linear thermal source in the test sample. Unlike the previous method, the heat source has a relatively large dimension and in this case, if the tested material is solid, the minimizing of thermal contact resistance between the probe and the material tested is an additional difficulty. Therefore, the hot wire method is rather used for thermal conductivity measurements in liquids or bulk solids26. The another method used in thermal conductivity research is the steady state technique—guarded hot plate method (GHP)—when material sample that is analyzed is in complete thermal equilibrium. The guarded hot plate method is widely used in measurements of thermal conductivity of insulation materials, that materials characterized by high thermal resistance. Examples of recent works are29. The rGO/rubber composites, being the subject of the present work, are also characterized by high thermal resistance. Generally, the rubber—matrix of composites tested—is a poor heat conductor. It can be expected that the addition of graphene oxide will not cause very significant changes in thermal conductivity. This has been confirmed in preliminary studies. Thus it seems that the guarded hot plate method is right in rGO/rubber composites research. An additional advantage is the method accuracy. It is in the range from 3 for 8%. In the present research the authors determined the expanded uncertainty of thermal conductivity at about 6%. Comparing with the another technique—the laser flash method—an accuracy of the GHP method will be better. In the case of laser flash method the uncertainty of thermal diffusivity, specific heat and density measurements increase the uncertainty of thermal conductivity measurement.The literature reviewing sees that nowadays, in the measurements of thermal conductivity of rubber composites, the researches use techniques: laser flash method3.The GHP has been used for measurements of graphene oxide/rubber composite thermal conductivity also by the other researchersδ = 7.65 mm for N0, N1, N2 and δ = 7.67 mm for H0, H1, F1, F2; d = 50.8 mm for all samples. It has ensured obtaining the averaged values of thermal conductivity in the entire volume of the sample. Additionally, the thermal contact resistance between test sample and the apparatus plates  could be easily minimized. This is possible thanks to apply a compressive load on the test stack consisting of material sample and the heating and cooling plates.In the rGO/rubber composites considered in the present study, the distribution of reduced graphene oxide flakes is not uniform. Agglomerates are present in the tested material, thus, for the obtain reliable results, a relatively large material sample should be tested. Dimensions of the circular samples investigated were: 30. The instrument applied is commonly named as a guarded hot plate apparatus (GHP). Figures Thermal conductivity of the rGO/rubber composites with different graphene oxide contents was measured by the guarded heat flow meter method with the use of the Unitherm 2022 thermal conductivity apparatus. Measurements have been performed according to the Standard31.The GHP technique is widely taken as a primary method to determine the effective thermal conductivities of materials with relatively high thermal resistance. The GHP apparatus applied was consisted of a flat-plate test sample, a hot-surface assembly and a cold-surface one. The cold and hot surface assemblies have been placed above and below the test sample, respectively. The thermal interaction of the apparatus with the surroundings was prevented by the insulation material. The test sample of the composite material was held under a reproducible compressive load between two metal surfaces, each controlled at a different temperature. The lower contact surface was a part of a calibrated heat flux transducer. During the heat flow from the upper surface through the composite material to the lower surface, an axial temperature gradient was established. The measurement of the temperature difference across the sample along with the output from the heat flux transducer and the known sample thickness enabled determining thermal conductivity of the composites investigatedThe thermal resistance of a composite material sample has been obtained using Fourier’s law in a steady state. Assuming thermal equilibrium the Fourier’s law applied becomesRs—thermal resistance of the composite material sample, Tu—upper plate surface temperature, Tm—lower plate surface temperature, q—heat flux through the sample, Rint—total interface resistance between sample and surface plates.δ is a thickness of the sample tested.In turn thermal resistance is defined asq has been determined by measuring the appropriate temperature difference across the reference calorimeter according toTL is the bottom heater temperature and N is the reference calorimeter heat transfer coefficient.The heat flux q obtaining and the value of resistance Rint in Eq. /2 was in the range 25–26 °C. Temperature measurements have been performed with the use of the system of thermocouples type K which was the element of the applied GHP apparatus. The uncertainty of the temperature measurement method was equal to 2% while the uncertainty of the GHP method for thermal conductivity evaluation was 3%. Expanded uncertainties of thermal conductivity and temperature measurements have been estimated according to33. They were about 6.5% and 4% for thermal conductivity and temperature respectively.As in the preliminary investigations the thermal conductivity measurements have been performed for the rGO/rubber composites included in Table As an example, the detailed results of four tests performed for N1 material sample are included in Table Measurement results for all composites tested with the use of GHP apparatus are summarized in Table The investigation results received show that the addition of the reduced graphene oxide into different rubber matrix causes an increase of thermal conductivity. The visible changes in thermal conductivity are rather small and they are in the range of measurement uncertainty. However, the preliminary investigations in which the thermal regular regime characterized by the constant samples temperature differences has been achieved, confirm the influence of rGO addition on thermal conductivity. The maximum difference is observed in the case of rGO/NBR composites: N0 and N2, and it is about 11%. This result is comparable to the preliminary tests basing on the thermal regular regime theory. In turn the HNBR results are the same for H0 and H1 samples. However, the expected increase in thermal conductivity depending on graphene oxide content may be within the measurement error. It is similar in the case of FKM, where the differences in thermal conductivity are in the order of 4%.34 (Maxwell–Eucken) is given by the effective thermal conductivity equation in the formOn the basis of the results received for rGO/rubber composites thermal conductivity of the reduced graphene oxide applied has been estimated. Two classical models of effective thermal conductivity of two-component materials have been used. The first—ME model34 (Effective Medium Theory) is given byThe second EMT modelkef represents thermal conductivity of the composite material, subscripts 1 and 2 represent continuous phase of the material and the dispersed phase, respectively.In both mentioned above models φ1 and φ2, thermal conductivity of the rGO – k2 has been calculated. Application of the model ME gave the result k2 = 34.3 W/(mK). In turn from the model EMT—k2 = 21.1 W/(mK). The results received are in a good accordance with the literature results35 obtained for the graphene oxide with the use of non-equilibrium molecular dynamics method.Basing on the thermal conductivity of the N0 and N1 samples, using the formulas  and 6)  6) and tThe use of classical structural models to determine thermal conductivity of rGO validated the thermal conductivity measurement method applied.In the present study, investigations of thermal properties of the reduced graphene oxide/rubber composites have been performed. As matrix materials NBR, HNBR and FKM rubbers have been used. The graphene oxide reduced with sodium hypophosphite has been applied as a filler material improving mechanical and thermal properties of the composite. The composites were investigated in terms of applications as a material of the roller bearing seals. Before thermal investigations the experimental research on some mechanical properties of the composites considered have been performed. The results of the investigations showed an improvement in elastic modulus and indentation hardness of the rGO/NBR and rGO/HNBR rubber composites with the increase of rGO concentration.Thermal conductivity of rGO/rubber composites increases with rGO concentration increasing, the maximum enhancement occurs in the case of NBR matrix material.Due to heterogeneity of the composites tested the measurement method used in investigations is very important. The size of the material sample is significant.The another important factor determining the correctness of results is taking into account the measurement uncertainty. In the present study the differences in some results received are in the range of the measurement uncertainty.Thermal conductivity results received with the use of guarded hot plate method are consistent with the results from comparatively method based on thermal regular regime. Thus, the results can be regarded as reliable.The improvement of the rGO/NBR thermal conductivity compared with the pure rubber is comparable with the literature results received by guarded hot plate method for the same graphene oxide content. The received compatibility concerns the results with taking into account measurement uncertainty.The use of classical structural models for two-component materials verifies the measurement method applied in the present work to determine thermal conductivity of rGO/rubber composites.Thermal conductivity of the composites has been measured with the use of guarded hot plate technique. Detailed calculations of the measurement uncertainty of the method applied have been carried out. The preliminary qualitative investigations by comparatively method have been also performed. SEM analysis has been made and it showed that the tested composite samples contain agglomerates of rGO nanoparticles. On the basis of the studies conducted and the obtained results some conclusions can be formulated:


Chemical reaction with diazonium molecules has revealed to be a powerful method for the surface chemical modification of graphite, carbon nanotubes and recently also of graphene. Graphene electronic structure modification using diazonium molecules is strongly influenced by graphene growth and by the supporting materials. Here, carrying on a detailed study of core levels and valence band photoemission measurements, we are able to reconstruct the interface chemistry of trimethoxybenzenediazonium-based molecules electrochemically grafted on graphene on copper. The band energy alignment at the molecule-graphene interface has been traced revealing the energy position of the HOMO band with respect to the Fermi level. In pristine graphene, in fact, the Fermi energy separates the occupied and empty states at the Dirac point thus making it a gapless semimetal with a low density of states at the Fermi level. Due to the small conductivity of graphene in this condition, various approaches are being explored to change the charge carrier concentration such as the direct doping of graphene through the chemical modification of graphene itself, either by the introduction of defects or by elemental substitution of the carbon or through molecular adsorption. At the same time, an electronic bandgap for graphene, mandatory to develop graphene-based electronic devices, can also emerge as a result of the interaction with the substrate or the introduction of atomic doping, as well as of the presence of organic molecules on the graphene layer7. Nowadays, methods of graphene chemical modification includes the covalent attachment (or grafting) of aryl groups onto the graphene surface, which transforms the sp2 carbon atoms into the sp3 hybridization state9. With respect to the adsorption  of the organic molecules on the graphene layer, the functionalization by covalent bonding is efficient in the graphene electronic structure modification1.Being simple and scalable, chemical modification is becoming a promising approach to modify the graphene electronic structure2 carbon atoms of graphene forming a covalent bond. The reduction reaction consists of an electron transfer to the aryl diazonium ions, releasing molecular nitrogen and creating a reactive intermediate aryl radical. This radical usually reacts directly with the carbon surface or can undergo polymerization with other aryl radicals. Electron transfer to diazonium ions can be spontaneous or can be controlled electrochemically . To date, a lot of different diazonium salts have been used for graphene modification9.Based on the previous experimental and theoretical experiences with fullerene and carbon nanotubes,  reduction of diazonium ions shows to produce highly reactive free radicals which attack the spThe Gr/Cu (graphene/Cu) interface is extensively studied and is among the candidates for scaling up the production of graphene. In this context the evaluation of the energy level alignment between the organic layer and graphene is of paramount importance to develop hybrid junctions. This feature makes the overall system interesting to develop devices based on a metal/Gr/organic layer interface where the covalently bonded diazonium-derived molecules can interact with the environment and transfer charges to the graphene layer beneath.It is important to note that the covalent attachment of aryl groups onto the graphene surface requires the use of an electrolyte solution, containing acids and salts, that makes this technique potentially exposed to contaminations that could in principle affect the properties of graphene. In order to assess the consequences of electrochemically assisted grafting on the graphene electronic structure, a systematic study of the electronic properties across the different stages of sample preparation and grafting is mandatory.in situ generated 3,4,5-trimethoxybenzenediazonium (TMeOD) cations. This compound is expected to form aryl radicals via electrochemical reduction and to graft the graphene as 3,4,5-trimethoxyphenyl (TMeOP) units10  cation, leading to the grafting of 3,5-bis-tert-butylphenyl (TBP) units, as well as from grafted Highly Oriented Pyrolytic Graphite (HOPG). TBD has been chosen as it does not contain oxygen, allowing for an unambiguous discussion of the oxygen contribution to the measured spectra. Indeed, oxygen may have a three-fold origin, i.e. from the growth solution (water and NO2), from unreacted TMeOP, but also from the Gr/Cu interface. Finally, the band structure at the Γ and K points of the Brillouin zone has been experimentally probed by angle-resolved photoemission spectroscopy (ARPES) with synchrotron radiation. Supported by density functional calculations on 3,4,5-trimethoxybenzene, as well as on TMeOP-grafted on free-standing graphene the analysis of the density of states in the valence band region has also allowed us to identify the HOMO band of TMeOD and set its energy with respect to the Fermi level, thus obtaining a scheme for the band alignment at the Gr/TMeOP hybrid interface.The chemically modified graphene has been characterized through Raman spectroscopy. Then a detailed study of both core levels and valence band has been carried out by comparing the photoemission spectra collected from the pristine, from the treated and from TMeOP-grafted graphene. In order to single out the contribution of the grafted molecular units to the core-level photoemission spectra, the data of the C 1 s, O 1 s, and N 1 s core levels have been contrasted and compared to those collected from graphene exposed to −1, is associated with in plane C-C stretching of sp2 hybridized carbon atoms in the planar graphene skeleton15. The 2D peak is associated with in plane breathing mode of carbon atoms and is a second order Raman scattering process  groups of the TMeOP units are substituted by C4H9 groups where the oxygen is missing, allowing to better single out the different contributions in the carbon and oxygen core levels.After the grafting procedure, core level photoemission spectroscopy measurements were carried out using X-ray photoelectron spectroscopy (XPS) (a) on the pristine graphene on Cu foil (Gr/Cu), (b) on the treated Gr/Cu foil, and (c) on Gr/Cu foil grafted with TMeOP units. XPS spectra have been collected also on (d) 5 mM of TBP-grafted graphene on Cu foil for comparison. The TBD units has been chosen because, as shown in Supplementary Information, the methoxy  and they can be ultimately found anchored to the low sp2 defects of graphene lattice created by the electrochemical process or they can intercalate between graphene and copper.The spectrum of the N 1 s core level can be interpolated by two Lorentzian peaks ascribed to the amine nitrogen group (N-H) at 399.5 eV2 bonding24.The C 1 s spectra are shown in Fig. 9).In addition, other structures appear at larger BE for the treated sample. Two features can be ascribed to the graphene π−π* satellite and to the oxygen contamination (O-C=O contribution3 feature) in the C 1 s confirms the success of the EC grafting procedure and suggests that the graphene coverage by TMeOP molecules is very high. We estimate, as shown in the supplementary, a coverage of about 1 molecule per 3 graphene unit cells. This result is in agreement with the increase of the D peak in the Raman spectrum if we consider the high density defect regime25 ascribed to the grafted molecules. In this approximation, we obtained, in fact, LD=1.07 nm which corresponds to a molecular density of 1 molecule per 4.5 ± 0.5 graphene unit cells in agreement with the coverage estimated by XPS measurements.For the TMeOP-grafted Gr/Cu foil, the presence of the methoxy contribution  are related to the graphene surface contamination due to the air exposure and it is generally ascribed to C-O and H2O groups. The electrochemical process seems to mainly eliminate these oxygen contamination components. This result is in agreement with the literature27, where the electrochemical process is usually adopted to reduce the graphene oxide. Traces of this contamination persist in the treated and grafted samples as proved by the presence of the O-C=O feature.The analysis of oxygen is more complicated Fig. . The eff30.The contribution, at lower binding energy (at about 530.5 eV) in all the samples can be ascribed to the oxygen trapped between the graphene and the copper substrateTo confirm this interpretation, we have verified that when the XPS measurement is more surface sensitive, by changing the analyzer take-off angle from the normal emission  to the take-off angle of 30° (more surface sensitive), the contribution of the oxygen contamination doubles  groups. As shown in the supplementary information  for pristine Gr/Cu and TMeOP-grafted on Gr/Cu at different molecular concentrations Fig. . In the  eV Fig. . When gr eV Fig. . The lac eV Fig. . It is kThe ARPES spectra on the 5 mM TMeOP-grafted Gr/Cu foil Fig.  at the ΓIn order to understand the effects of TMeOP grafting on graphene, we carried out a set of several ab-initio simulations. At first, we considered the isolated 3,4,5-trimethoxybenzene (TMeOB), as shown in Fig. 2.Then, free-standing graphene and TMeOP-grafted graphene, have been considered Fig. . In thes3 C atom and its three C neighbours are lifted of about 0.581 Å and 0.174 Å, respectively. Figure The grafting induces structural and electronic modifications. The graphene is strongly buckled with respect to the unrelaxed, free-standing, graphene plane the sp2 to sp3 at the grafting site. In fact, consistent with literature35, a change in carbon hybridization from sp2 to sp3 in the graphene layer introduces a flat band at the Fermi level, yielding a small peak in the DOS. This state is not present in ARPES measurements. We ascribe this discrepancy to disorder effects that are not accounted for in the supercell calculations. The DFT calculations are in fact carried out considering an ordered structure formed by a TMeOP units bound over a 5 × 5 graphene supercell, as shown in Fig. We note that the electronic structure calculations predict a weak peak (B) in the density of states (DOS) at the Fermi level. The origin of this in-gap state is mainly due to the breakdown of the lattice symmetry of graphene when grafted by TMeOP units and to the change in carbon hybridization from spx/Cu. The other DOS feature ascribable to graphene, peak A in Fig. In spite of the electronic hybridization between the molecule and graphene, it is possible to easily pinpoint the TMeOB contribution in the integral density of states (DOS) spectra, shown in Fig. The interface chemistry of graphene grown on copper and functionalized by covalent bonding with TMeOP units has been studied by core level and angle-resolved valence band photoemission. Two main effects can be ascribed to the electrochemical treatment, the application of the electric field creates a low density of defects in graphene that can accommodate N-groups, in addition the treatment eliminates the oxygen contamination on the top of the graphene layer.x layer between graphene and copper allows to regard graphene as almost decoupled from the metallic substrate and to measure the alignment between graphene σ and π bands and the HOMO of TMeOP, which is found to lay about 1 eV above the top of the σ bands, consistently with DFT calculations.Moreover, comparing the core level spectra collected on both TMeOP and TBP-grafted on graphene, the contribution of the methoxy groups has been singled out in C 1 s and O 1 s spectra. An extra oxygen contribution coming from a CuOTherefore, despite the presence of the solution in the electrochemical cell, that makes this treatment a grafting procedure potentially exposed to contaminations from electrolytes, the results reported in this study show that the functionalization by diazonium chemistry does not affect significantly the chemical environment, and that the electronic properties at the interface are mainly determined by the covalently grafted molecules.36, therefore disclosing the possibility to apply photoemission techniques to systems prepared on the basis of similar electrochemical methods37, and ultimately enabling a scalable pathway for the production of functionalized graphene layers.This finding demonstrates that high quality grafting can be achieved in a set-up with growth conditions less demanding with respect to UHV-related methods2, Pt wire counter and Ag/AgCl/3 M NaCl reference electrodes. 3,5-bis-tert-butylbenzenediazonium (TBD) chloride and 3,4,5-trimethoxybenzenediazonium (TMeOD) chloride are unstable and decompose rapidly: hence they were synthesized from the corresponding aniline precursor immediately prior to electrochemical reduction. This procedure involves 5 mL of a 1 mM (or 5 mM) 3,4,5-trimethoxyaniline  or 3,5-bis-tert-butylaniline  in 50 mM HCl (Sigma-Aldrich) aqueous solution which was mixed with 50 µL (or 250 µL) of aqueous NaNO2 (0.1 M) for activation of the diazotization reaction. Within 90 seconds, this mixture was gently shaken and pipetted into the EC cell. Cyclic voltammetry was used for the electrochemical activation. In the measurements the potential window was chosen from 0.3 V to −0.6 V, scan rate 0.1 V/s for 3 cycles. After modification, the TMeOP modified samples were rinsed with Milli-Q water  to remove any physisorbed material from the surface and dried in a stream of Argon. The STM measurements were acquired with a molecular imaging STM system operating in constant current mode. The tip was obtained by a Pt-Ir wire .Electrochemical measurements were performed using an Autolab PGSTAT101 potentiostat . The electrochemical modification procedure was carried out in a homemade single-compartment three–electrode cell with a working electrode area of 38.5 mmThe substrates used are CVD graphene on Cu foil obtained from Graphenea, graphene on Cu(111) obtained from IMEC and HOPG .38 VG-SCIENTA R3000 analyzer and the Al Kα line of a twin anode X-ray source, operating in ultra-high vacuum conditions (base pressure 2×10−10 torr).In order to avoid the detachment of the phenyl units, all the X-ray photoemission measurements have been collected after an annealing treatment in ultra-high vacuum conditions at temperatures lower than 393 K. Core level spectra have been collected with a properly calibrated39. The overall energy and angular resolution were set to 100 meV and 0.3°, respectively. All the ARPES maps were collected at room temperature (RT) after an annealing treatment. The annealing temperature , in this case, is the minimum value that allows to observe a clear ARPES map.Angle-resolved valence band spectra were carried out at the BaDElPh beamline of the Elettra synchrotron in Trieste  using a photon energy of 34 eV and an hemispherical electron analyzer SPECS Phoibos 150 with a 2D-CCD detector system2. Raman scattering was collected with the same objective and directed to a Raman spectrograph  equipped with a cooled-charge coupled device (CCD) camera operated at −100 °C  through a pinhole, a dichroic mirror  and long pass filter . Accumulation time for each point in ‘grating’ measurement was 1 s. All of the measurements were carried out under ambient conditions and at room temperature.Raman measurements were performed with an OmegaScope 1000 (AIST-NT). Laser light (632.8 nm) from a He-Ne laser was focused onto the sample surface from the side (with an angle of 28° to sample surface) and top, for ‘grating’ and ‘normal’ measurements, respectively, through an objective . Optical density at sample surface was about 500 kW/cm40. Van der Waals corrections to the exchange-correlation potential have been neglected, due to the direct chemical bond induced by grafting. Calculations have been performed with the ABINIT package41 in the framework of Projector-Augmented wave atomic description. A large cut-off energy (700 eV) has been considered for the plane-wave basis definition. All atomic position have been relaxed up to a maximal interatomic force of 10–5 Ha/Bohr. For the graphene and the grafted graphene cells a 7 ×7 ×1 Monkhorts-Pack grid has been adopted, with an adequately large cell size on the out-of-plane direction (15 Å); for the isolated TMeOB, the molecule was placed in a wide (15 Å) cubic cell size, with a single k point. For each case, the convergence on the total energy was set to the 10−9 Ha level.The TMeOB, TMeOD-grafted graphene and pristine graphene electronic structures have been evaluated through ab-initio density functional theory calculations, in the framework of the GGA-PBE approximationSupplementary information.


The side electrode structure is designed to reduce the mode loss induced by the graphene film and by heating the electrode. The graphene layer is designed to be located on the waveguide to assist in the conduction of heat produced by the electrode. The inverted ridge core is fabricated by etching and spin-coating processes, which can realize the flat surface waveguide. This core improves the transfer of the graphene layer and the compatibility of the fabrication processes. Because of the opposite thermal optical coefficient of polymer and silica and the high thermal conductivity of the graphene layer, the 3D hybrid TO switch with low power consumption and fast response time is obtained. Compared with the traditional TO switch without graphene film, the power consumption of the proposed TO switch is reduced by 41.43% at the wavelength of 1550 nm, width of the core layer (a) of 3 μm, and electrode distance (d) of 4 μm. The rise and fall times of the proposed TO switch are simulated to be 64.5 μs and 175 μs with a  Three-dimensional 3D) hybrid integrated chips have the advantages of a high integration degree of photonic chips, improvement and diversification in device function, and low signal crosstalk; consequently, these chips can be applied to optoelectronic integration fields and 3D integration fields ,2. A varD hybrid A key goal of 3D hybrid integrated optical devices is to realize the optical path switching between different layers. Therefore, the X-junction vertical structure optical switch has been proposed ,16,17. ITo further reduce the power consumption of the vertical optical devices, the polymer/silica hybrid MZI TO switches with a graphene film have attracted attention. Two-dimensional graphene material has many excellent optical properties such as large thermal conductivity, high carrier mobility, and wide optical response spectrum . TherefoIn this paper, a 2 × 2 MZI-inverted ridge TO switch of a graphene–polymer–silica hybrid waveguide with side electrode structure is proposed. The waveguide grooves are designed to be fabricated by the etching process. Then, the polymer core layer was obtained by polymer spin-coating in grooves. This process can realize the compatibility of silica and polymer materials and the flat surface waveguide, thus helping in the transfer of the graphene film. Because of the high thermal conductivity of the graphene layer and the opposite thermal optical coefficient of polymer and silica, the 3D-inverted ridge TO switch of the graphene–polymer–silica hybrid waveguide with low power consumption and fast response time can be obtained.The 3D structure of the MZI-inverted ridge 3D hybrid TO switch with a graphene film is shown in a = b = 3 μm. The parameters of m = 6 μm, eL = 1 cm, and 2d = 0.6 μm indicate the width of the electrode, length of the electrode, and coupling gap between two waveguide core layers, respectively. The thickness of the slab layer is h = 1.22 μm. The coupling length of the waveguide in the vertical direction is cL. The horizontal distance between the center of the core layer and the center of the electrode is d. cn1 = 1.48, cn2 = 1.478, and n1 = 1.444 are the refractive indices of the silica core layer, polymer core layer, and silica cladding layer, respectively. When the effective refractive index of the two core layers is equal, the optical field can be theoretically fully coupled in a 3-dB coupler. When heating the electrode, the effective refractive index of the waveguide changed, realizing the phase change of π.The dimensions of the core layers are −4/°C and 1.28 × 10−5/°C. The phase change ∆Φ can be expressed as:neff is the effective refractive index change, and λ is the wavelength of waveguide (set at 1550 nm). Because of the large thermal conductivity of a graphene layer , the graphene layer is coated on the waveguide to assist the conduction of heat produced by the electrode. This process helps realize the phase shift at a lower power consumption (P) of the 3D hybrid TO switch.The thermo-optical coefficients of the polymer and silica are −2.36 × 102n is the refractive index of the polymer cladding layer. To realize the complete coupling of light field, the structure of the waveguide has been optimized . a = b = 3 μm; c1n = 1.48; cn2 = 1.478; n1 =1.444; h =1.22 μm; d = 4 μm). The optical field distribution and the thermal field distribution of the devices are simulated by finite element analysis software. The simulation processes include geometry build, add material, physics analysis, meshing build, study, and results analysis. For the optical field analysis, the physics interfaces are “electromagnetic waves” and “frequency domain”. The meshing method and the study method are “physics-controlled mesh” and “mode analysis”, respectively. For the thermal field analysis, the physics interface is “heat transfer in solids”. The meshing method and the study method are “free triangular mesh” and “stationary”, respectively. First, the top electrode structure of the TO switch by lithography process has been simulated  induced by graphene film and electrode as a function of horizontal distances (d) between the center of the core layer and the electrode with the electrode width (a) from 1.5 to 3.5 μm. Five different polymer core layer dimensions are designed by COMSOL, realizing the complete coupling between two waveguide core layers. Because of the absorption of light by the side electrode, as shown in d > 4 μm).a = 3 μm and a = 2 μm, respectively. For the TO switch without a graphene film structure, the power consumption significantly increases as a function of electrode distance (d). The power consumption is 3.2 mW with a d of 0 μm, while the power consumption increases to 17.26 mW with a d of 7 μm. For the graphene TO switch structure, the power consumption is insensitive to the electrode distance because of the large thermal conductivity of graphene film, relaxing the tolerances during the fabrication work of the devices. The results show that the power consumption for the graphene TO switch with d of 4 μm is reduced by 23.13% compared with the top electrode TO switch structure (d = 0 μm) without graphene film.d = 4 μm) are shown in The calculated response time and the power consumption with different electrode widths (d of 4 μm, a of 2 μm, ∆T of 1.15 K, and P of 2.95 mW, respectively.d of 4 μm, a of 3 μm, electrode length of 1 cm, and wavelength of 1550 nm. Compared with the inverted ridge TO switch with a top electrode structure, the power consumption was reduced by 23.13% for the graphene TO switch with a d of 4 μm. Moreover, the rise and fall times were 64.5 μs and 175 with a d of 4 μm, a of 2 μm, ∆T of 1.15 K, and P of 2.95 mW, respectively.In summary, we have presented an inverted ridge 3D hybrid TO switch with graphene film. Because of the opposite thermal optical coefficients of polymer and silica and the high thermal conductivity of a graphene layer, a graphene-based side electrode TO switch with low power consumption and fast response time has been obtained. Compared with the traditional 3D polymer–silica hybrid TO switch with top electrode structure, the power consumption was reduced by 41.43% for the graphene TO switch with a


However, no differences were observed between the total doses of corticosteroids administered over days 31 to 60, 61 to 90, 91 to 120, and 151 to 180 after the AE. Furthermore, differences between the administration rates of immunosuppressive and antifibrotic treatments administered to the 2 patient groups were not significant. An increased total dose of corticosteroid administered over day 1 to 30 after an AE-IPF was associated with a decreased risk of subsequent recurrence of AE-IPF within 1 year after the first AE.Some patients with idiopathic pulmonary fibrosis (IPF) undergo recurrent acute exacerbations (AEs). This study aimed to elucidate the risk factors for recurrent AEs of IPF (AE-IPF). Consecutive patients with IPF admitted for their first AE-IPF between January 2008 and December 2018 were retrospectively recruited. Of 63 patients admitted for an AE-IPF and discharged alive, 9 (14.3%) developed a recurrence of AE within 1 year. The mean time to recurrence was 233 ± 103 days. Total doses (mg/month and mg/kg/month) of corticosteroids administered over day 1 to 30 after the AE were significantly higher in patients without recurrences of AE-IPF  than the doses in patients with recurrences  ( Over their clinical course, some IPF patients undergo rapid worsening of respiratory symptoms associated with new ground-glass opacities or consolidations on chest high-resolution computed tomography (HRCT). The event is termed “acute exacerbation (AE) of IPF” (AE-IPF), and its mortality rate is high2. The treatment for AE-IPF generally consists of high-dose corticosteroids and/or immunosuppressive agents, although there have been no supportive data from randomized controlled trials4. There is also no proven specific maintenance treatment after the initial treatment.Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrosing interstitial pneumonia of unknown etiology5. However, specific risk factors for the subsequent recurrence of an AE in patients after their first AE remain unknown. The aim of this study was to test the hypothesis that as one of the treatments, the initial dosage of corticosteroid for the first AE is associated with early recurrence of an AE-IPF.AE-IPF sometimes recurs, even after it has resolved. A previous history of AE was reported to be associated with an increased risk of recurrenceDuring the study period, 86 patients with IPF were hospitalized for AE Fig. . Among tThe baseline clinical characteristics and treatments of the 63 patients admitted for the first time and discharged alive are shown in Table p = 0.02 and p = 0.03, respectively). However, no differences were observed between the total doses of corticosteroids administered over days 31 to 60, 61 to 90, 91 to 120, and 151 to 180 after the AE. Furthermore, differences between the rates of administration of immunosuppressive and antifibrotic treatments to the 2 patient groups were not significant. A multivariable analysis of the following independent variables: total doses (mg/month and mg/kg/month) of corticosteroids over days 1 to 30 after the diagnosis of AE, total doses (mg/month) of corticosteroids over days 61 to 90, sex, age, BMI, and use of antifibrotic agent at discharge; identified total doses (mg/month) of corticosteroids over days 1 to 30 as the only independent predictor of recurrence .Treatments for the first AE-IPF are shown in Table This study found that the total dose of corticosteroids over days 1 to 30 after the diagnosis of AE-IPF were associated with subsequent recurrence(s) of AE-IPF within 1 year after the date of admission for the first AE-IPF. A treatment that combined steroids with another immunosuppressive agent was not associated with recurrent AE. Furthermore, antifibrotic therapy administered within 1 year after AE-IPF was not associated with a subsequent recurrence. To our knowledge, this is the first study which has shown a significant association between the dose of corticosteroid used to treat a first AE-IPF and the subsequent recurrence of AE.6. However, the initial dose of corticosteroid for an AE-IPF is usually reduced gradually because of concern about a recurrence. Our study found that only the initial dose of a corticosteroid during the first month after the onset of an AE-IPF was important in regard to preventing a recurrence of an AE. It might be possible that the initial dose of a corticosteroid can be reduced rapidly after the first month of treatment for the AE-IPF. If this can be verified, the frequency and severity of side effects associated with corticosteroid therapy could be markedly reduced.Corticosteroids often cause serious side effects, especially at high doses administered over a long period of timeAn immunosuppressive agent tended to be more frequently used in addition to a corticosteroid for the patients who did not develop a recurrence of AE-IPF than for those who did develop a recurrence (11.1% use by patients with vs. 35.1% use by patients without), although statistical significance was not achieved. An immunosuppressive agent might be a possible treatment option not only for treating patients with an AE-IPF, but also for preventing recurrence of an AE, although further study is needed to test this hypothesis.7. Pirfenidon has also been reported to reduce the incidence of AE after surgical resection for patients with IPF and lung cancer8. In our study, however, antifibrotic therapies given within 1 year after the first AE-IPF were not associated with the recurrence of an AE-IPF. Antifibrotic agents have been usually started in our institution after the patient has stabilized. Given this practice for administration of antifibrotic agents, the duration of antifibrotic therapy given to some of the study patients over the year of observation after the diagnosis of an AE-IPF might have been too short. If the observation period had been longer than 1 year, a different result might have been observed. Another possible explanation for our finding is that because a small number of patients received the agents, the effects of antifibrotics were missed. The efficacy of antifibrotic agents for reducing the risk of a recurrent AE-IPF should be investigated further.The antifibrotic agents nintedanib and pirfenidone are approved drugs for the treatment of IPF. Nintedanib was reported to reduce the risk of AE-IPF in an efficacy study of patients with IPF9. Among the 7, two (28.6%) had a history of 3 episodes of AE-IPF. The recurrences reportedly occurred in most patients over a short period of time  after the previous AE. In our present study, the mean time to recurrence of an AE-IPF was 7.7 (range 2–12) months, which was similar to the previous report. There may be a phenotypic patient with IPF who develops frequent exacerbations.Another important finding of the study is that a recurrence of AE-IPF is also a risk factor for the subsequent recurrence of an AE. In this study, of the patients developing a recurrence of AE-IPF after their first AE, 42.8% developed a second recurrence. This result is similar to that of the previous study, which demonstrated that of 27 patients with an AE-IPF, 7 (25.9%) had more than 1 episode of AE4. A triggered AE includes an AE occurring after an infection, procedure, or surgery; or an AE associated with drug toxicity or aspiration. Distinguishing between infection-triggered AE and simple pulmonary infection is sometimes impossible. Fourth, data at the discharge from the chest HRCT, pulmonary function testing, the six-minute walk test, and echocardiography could not be used for the analysis, because they were not performed in most cases.This study has limitations. First, this was a single-center study with a relatively small number of patients. Second, the study was conducted in a retrospective fashion. A multicenter study employing prospective enrollment of a larger number of patients is needed to confirm the results of our small study. Third, our study might have included patients with pulmonary infections. Major efforts were made to exclude patients with obvious pulmonary infections. However, distinguishing clearly between an AE-IPF and pulmonary infection is sometimes quite difficult. The recent international working group report of the American Thoracic Society (ATS) proposed 2 types of AE-IPF, idiopathic versus triggered AE-IPFIn conclusion, when comparing IPF patients hospitalized for their first AE, which was treated by steroids, who were stratified according to whether or not they developed an early subsequent recurrence of an AE, a higher total dose of corticosteroids administered over days 1 to 30 after admission was associated with a lower risk of early recurrence of a subsequent AE-IPF.4. The diagnosis of IPF was made according to the criteria used in the INPULSIS trial7. If a surgical lung biopsy was not performed, a HRCT study was required, which showed the following findings: honeycombing and/or a combination of reticular abnormalities and traction bronchiectasis consistent with fibrosis with basal and peripheral predominance and without atypical features of UIP7. The study protocol was approved by the ethics committee of the Kindai University Faculty of Medicine (No. 31-228). Informed consent was waived, because this study was based on a retrospective analysis of case records from our university hospital. All methods were performed in accordance with the relevant guidelines and regulations (Declaration of Helsinki).A retrospective review was performed of the files of consecutive patients with IPF who were admitted to the Kindai University Hospital for their first AE between January 2008 and December 2018 and were discharged alive. Whether or not the patient developed a recurrent AE within 1 year from the date of onset of the first AE was determined. The diagnosis of AE-IPF was made according to the recent international working group report of the ATS, as follows: 1) a previous or concurrent diagnosis of IPF; 2) acute worsening or development of dyspnea typically of less than 1-month’s duration; 3) computed tomography study of the lungs showing new bilateral ground-glass opacities and/or consolidations superimposed on a background pattern consistent with UIP; 4) deterioration not fully explained by cardiac failure or fluid overload11.Pulmonary function test results that were obtained during the stable phase of the disease within 1 year prior to the AE were adopted as baseline values. Pulmonary function testing was performed by the CHESTAC-8800 system , according to the standards proposed by the European Respiratory SocietyThe following baseline characteristics of the patients with AE-IPF were determined, as follows: gender, age, body mass index, smoking status, long-term oxygen therapy, and treatments for IPF. Admission and discharge results of routine blood tests were evaluated.The total dose and the total dose per body weight (kg) of corticosteroids administered over each month after the initiation of steroid therapy were evaluated. Whether or not immunosuppressive agents were used with corticosteroids was assessed. Use of an antifibrotic agent within 1 year after the day of admission for the first AE was also examined.The recurrence of an AE-IPF within 1 year after the date of admission for the first AE-IPF was reviewed in each patient. The time to recurrence was defined as the number of days from the date of admission for the first AE-IPF until the date of admission for the recurrent AE.t-test was used for continuous data and the χ2 test or Fisher exact test was used for categorical data between 2 groups. Multivariable analysis with logistic regression was used to identify the risk of AE-IPF recurrence. In this analysis, variables with p < 0.2 on univariate analysis and variables historically considered to be important in IPF were included as independent variables in the multivariable analysis. A p value of < 0.05 was considered statistically significant. Analyses were performed with Statflex, ver.6 software .Continuous variables were expressed as means ± standard deviation (SD), and categorical variables were expressed as actual numbers. The unpaired


Edge mode could disturb the ultra-subtle mass detection for graphene resonators. Herein, classical molecular dynamics simulations are performed to investigate the effect of edge mode on mass sensing for a doubly clamped strained graphene resonator. Compared with the fundamental mode, the localized vibration of edge mode shows a lower frequency with a constant frequency gap of 32.6 GHz, despite the mutable inner stress ranging from 10 to 50 GPa. Furthermore, the resonant frequency of edge mode is found to be insensitive to centrally located adsorbed mass, while the frequency of the fundamental mode decreases linearly with increasing adsorbates. Thus, a mass determination method using the difference of these two modes is proposed to reduce interferences for robust mass measurement. Moreover, molecular dynamics simulations demonstrate that a stronger prestress or a higher width–length ratio of about 0.8 could increase the low-quality factor induced by edge mode, thus improving the performance in mass sensing for graphene resonators. Since Geim and co-workers fabricated monolayer graphene by mechanical exfoliation in 2004 , graphen−24 g) with a mass responsivity of 0.34 GHz/yg. Then, Jiang et al. [To date, a number of authors have studied various methods to realize atomic-scale mass sensing by using graphene resonators. For example, Duan et al.  adopted g et al.  investigg et al. , utilizig et al. , and adjg et al. , have beg et al. , has rarThe molecular dynamics method, which directly computes the state of every atom and takes the scale effect into consideration, seems to be the most suitable method to investigate the edge mode of graphene sheets compared with finite element methods , nonlocaIn this paper, molecular dynamics simulation is performed to investigate the distinct responses between edge modes and fundamental modes to adsorbed mass on graphene sheets, and a novel mass determination method based on the frequencies of these two modes is proposed, which is expected to diminish the interferences caused by mutable stress in graphene. In previous studies, the mass was determined by the fundamental frequency shift, which is hypersensitive to the stress in graphene in addition to the absorbed mass. However, it is difficult to control the stress in graphene accurately and steadily. For example, temperature fluctuation can induce distinct thermal expansions between graphene and silicon oxide substrate, thereby changing the graphene inner stress significantly . MoreoveMolecular dynamics simulations have been used to study the impact of edge modes on mass sensing for strained graphene resonators ,45. The In the present MD simulation, the modeling schematic diagram of a graphene-based resonant mass sensor is shown in In L is the length of the graphene sheet and σ is the axial tension in graphene. The equivalent density ρ, in terms of the contributions from both graphene and adsorbates, is calculated considering the thickness of the monolayer graphene sheet to be 3.35 Å and the ideal density to be 2200 kg/m3 for pure grapheme [Referring to the previous literature ,6,53, doAs for the doubly clamped vibrating graphene sheet with a length of 100 Å and a width of 50 Å, as shown in To identify which vibration eigenmode the first peek represents, the mechanical vibrations of the graphene sheet were observed by visualization software OVITO , and notρ will increases, thus reducing the fundamental frequency. The frequency shift can be written byρ0 is the density of pure graphene and ∆ρ is the increment of equivalent density, assuming the absorbed mass is evenly distributed. The factor K is used to adjust the frequency shifts caused by distinct distributions of adsorbates and equals 1 when adsorbates are evenly distributed. Since the volume of graphene sheet can be regarded to be unchanged, Equation (2) is then rewritten asm0 is the mass of pure graphene sheets and ∆m represents the mass of adsorbates. In contrast, due to the unpredicted results when using standard elastic beam theory, the frequency of edge modes shows a considerably different response to central adsorbates.Resonant mass sensors are dependent on the fundamental frequency shifts in response to adsorbates. When the external mass is absorbed on the graphene sheet, the equivalent density 0.5 according to Equation (1), and it matches with the MD results considerably well, with an R-square of more than 0.99 and a maximum relative error of about 6%. Moreover, this relative error decreases to about 2% if a stronger tension (>15 GPa) is exerted, which can be explained by the fact that the stretched graphene sheet is closer to the ideal tensioned membrane structure. On the other hand, the two curves in biasC is a constant. Equation (4) is then used to fit the MD results, showing an excellent R-square value up to 0.99, as displayed in To compare the distinct response to the centrally distributed absorbates between edge modes and fundamental modes, 1–10 gold atoms as adsorbates were placed in the middle of the graphene sheet with a length of 100 Å and a width of 50 Å, along with the applied axial stress ranging from 10 to 50 GPa. The working principal of typical graphene-based resonant mass sensors complies with Equation 3), where the linear relationship between the fundamental frequency and adsorbate mass is critical for mass determination, assuming the stress σ in graphene is constant. However, the stress in graphene sheet is susceptible to numbers of factors, such as the temperature fluctuations , the var, where tm is absorbed mass; m0 is the mass of the graphene sheet (excluding fixed areas); biasC is a constant as illustrated before, which is irrelevant to the stress in graphene. Note that the above derivation is under the premise that the mass is centrally distributed; nevertheless these adsorbates cannot be located at the exact middle of graphene sheets. Thus, the acceptable range of the adsorbates where the mass determination method proposed above is applicable is worth discussing.The MD simulation results shown in To determine the appropriate range for centrally distributed adsorbates, two gold atoms as absorbed mass were moved from the center of graphene sheets to the edges along the width direction, and then the corresponding frequencies of the two modes were recorded and are depicted in edgef ≈ fundamentalf − 30. In this case, we cannot obtain two independent equations regarding absorbate mass and stress and solve them together like in the case of central distribution. That is, the frequency of edge modes cannot provide more independent and valid information for mass determination, so the mass determination method proposed above is not applicable for evenly distributed adsorbates.The discussions and conclusions above make sense only in the case of centrally distributed adsorbates. When adsorbed mass is evenly distributed, the frequencies of the fundamental mode and edge mode decrease synchronously, as can be seen in The quality factor (Q) is always an important issue for resonant sensors, and a higher Q factor means less energy dissipation per oscillation period and higher resolution for mass sensing ,56. The f is the fundamental frequency; t is duration of the traced vibration; A0 and tA are the amplitudes of fundamental oscillation at the beginning and end of simulation, respectively. In terms of Equation (6), the Q factors of these four samples are then calculated to be 1463, 2520, 2321 and 4405, respectively. It is apparent from these four charts that the graphene sheet with smallest width and stress has much more energy loss in oscillation, while that with the largest width and stress has the least energy loss, and its Q factor is three times bigger than that of the former. The intermediate two also clearly show less amplitude diminishing, with Q factors of 2520 and 2321, respectively. Incidentally, the ripples on the margins of these amplitude curves occur due to the incoherent mixing of vibrations of two modes with different frequencies, which is called the beating phenomenon. Comparing the four results, it can be seen that enlargement of the width and stress could effectively improve the Q factor of graphene resonators.To elucidate the edge effects on the Q factor for improving Q value, graphene sheets with different widths and inner stress were investigated by recording the diminishing amplitudes of fundamental vibrations that imply energy dissipations. For clarity, the impacts of the width and stress on the Q factor are therefore further studied individually. In conclusion, classical molecular dynamics simulations are adopted to investigate the impact of edge mode on mass sensing for a doubly clamped monolayer graphene resonator. The vibrations of edge mode were found to localize at the free edges and possess larger amplitude. Specifically, in the MD simulation for a 100 × 50 Å graphene sheet, the vibration amplitude of edge mode achieved 3.8 Å, nearly 2.5 times that of the fundamental mode. Moreover, the frequency gap of the two modes was calculated to be 32.6 GHz, which is irrelevant to the axial stress in graphene. Thus, considering that fundamental frequencies drop linearly with increasing centrally absorbed mass, while the frequencies of edge mode remain constant, a novel method of mass determination combining the two vibration modes was developed in order to diminish the interference of mutable stress in graphene sheets. More importantly, the MD results show that the vibration of edge mode broke the coherence of the mechanical fundamental oscillation, thus aggravating the energy dissipation and then resulting in a lower Q factor, which was as low as 1463 for the 100 Å × 50 Å graphene sheet with tensile stress of 10 GPa. However, increasing the stress in graphene can greatly increase the Q factor up to 7989 when an axial stress of 50 GPa is exerted. Moreover, appropriately widening the graphene sheets with a higher width–length ratio above 0.8 can double the Q factor, although excessive width does not contribute to an extremely high Q factor.


Observed contractile response indicated on effective SMCs stimulation mediated by graphene layer which constituted efficient cell to biomaterial interface.Tissue engineering allows to combine biomaterials and seeded cells to experimentally replace urinary bladder wall. The normal bladder wall however, includes branched neuronal network propagating signals which regulate urine storage and voiding. In this study we introduced a novel biocomposite built from amniotic membrane (Am) and graphene which created interface between cells and external stimuli replacing neuronal network. Graphene layers were transferred without modifying Am surface. Applied method allowed to preserve the unique bioactive characteristic of Am. Tissue engineered constructs composed from biocomposite seeded with smooth muscle cells (SMC) derived from porcine detrusor and porcine urothelial cells (UC) were used to evaluate properties of developed biomaterial. The presence of graphene layer significantly increased electrical conductivity of biocomposite. UCs and SMCs showed an organized growth pattern on graphene covered surfaces. Electrical filed stimulation (EFS) applied  Following this concept, a leading scenario aims to create a neobladder by combining different biomaterials with autologous urothelial and detrusor cells. These two components are expected to create a favourable environment for functional bladder wall regrowth. The biomaterial plays a pivotal role in this strategy as its biomechanical characteristic and bioactivity profile determine cellular events associated with regeneration2. Within the bladder wall, there is multilayer branched neuronal network delivering and propagating signaling from the central nervous system (CNS) to the detrusor smooth muscle layer. Synchronous initiation of action potentials mediates the wavelike contraction, gradually embracing the whole bladder increasing intravesical pressure and generating urine outflow3.The urinary bladder is a temporary storage reservoir for urine. Both continence and voiding phases are active processes involving detrusor contraction or relaxation controlled by peripheral and central nervous system. In addition, passive mechanical properties of the bladder wall, such as viscoelastic force determine static and dynamic behavior of urinary bladder4. Nevertheless, none of them was successful in clinical translation due to the high complication rate and lack of long-lasting function. The major problem hampering advances in the field of urinary bladder tissue engineering is the simplification of the targeted neobladder structure in conducted research. Accordingly, one of the critical challenges hardly ever addressed in experimental settings was the restoration of the neuronal network within the neobladder wall5.To date, urinary bladder reconstruction with tissue engineering methods testing different usually acellular scaffolds has been used in 131 patientsDe novo regeneration of convolutional neural network within the neobladder hasn’t been ever tried due to lack of applicable technology. There might be however difrent ways to approach this dilemma. The alternative solution is to completely replace a native neural network with biocompatible current conductive material that might be linked to an external unit generating electrical stimulation6. However, before such a biocybernetics organ replacing unit may be created, there is a need to develop a biomaterial scaffold providing the interface between external stimuli and host tissue. Physiologically, the contraction of the detrusor muscle is initiated by meditators like acetylcholine and ATP (Adenosine Triphosphate) released from efferent nerve fibres. Subsequently triggered, discharge of the dispersing action potential is the direct signal contracting the smooth muscle layer7. On account of this two-stage mechanism, the chemical mediated stimulation could be replaced by direct electrical stimulation in the tissue-engineered bladder.8. Research data derived from in vitro and in vivo models indicated that application of electrical stimuli directly to detrusor muscle may trigger effective contraction. Nevertheless the major challenge is to spread and sustain locally induced depolarization wave9. In comparison to heart urinary bladder doesn’t have recognized intramural conduction system delivering electrical stimuli to all bladder regions10. Creation of tissue engineered bladder offer possibility to develop de novo conduction system within bladder wall intended to distribute electrical stimulation aimed to trigger controlled contraction pattern of reconstructed smooth muscle layer.Inducing detrusor contraction by direct electrical stimulation is a concept which has been developed to initiate micturition reflex in patients with neurogenic bladderIn this study, we are proposing a new biocomposite biomaterial derived from the amniotic membrane (Am) covered with a graphene layer. The developed method of graphene transfer allowed to cover intact Am with solid graphene layers. The primary object was to obtain an electrical stimuli-responsive biomaterial tailored for provisional application in the tissue-engineered urinary bladder. The ability to deliver electrical stimuli opens a new perspective for the generation of dynamic tissue systems demanding a functional and responsive smooth muscle layer.11. Its excellent bioactivity profile is mediated by active molecules incorporated within the Am matrix. Am was applied for experimental reconstruction of the bladder wall in rat model and proved its ability to support regeneration of smooth muscle layer12. Furthermore, Am has universal capability to induce epithelization including urothelial layer regrowth13. Knowing this, the overriding assumption during interdisciplinary development of graphene transfer method was to avoid any modification of the Am structure impairing its biocompatibility profile. At any stage of biocomposite fabrication Am didn’t have any contact with cytotoxic, cross-linking or oxidizing substances.Am is used in urological tissue engineering due to its unique properties supporting the regeneration process14. Therefore, an innovative strategy involving non-damaging graphene layer transfer on a biomaterial surface may overcome this limitation and become a novel solution worth further development and testing.Conductive biomaterials are of particular interests for tissue engineering, and recently many new biomaterials were introduced. Despite intensified research efforts, there are several significant limitations. The most substantial is reported to have reduced biocompatibility, and challenging necessity to combine hard or brittle conductive layer with softer and compliant natural derived biomaterial15. In this context, the application of graphene in regenerative medicine enables to cross biological barriers which are inaccessible to more abundant commonly evaluated materials. The most crucial advantage of graphene is a neglectable influence on passive biomechanical characteristics of the tissue-engineered bladder wall. Therefore it doesn’t impair the ability to disdain during repetitive cycles of urine storage and micturition. Moreover, graphene, due to its nanostructure, might reach single effector cells analogously to healthy neurons and provide adequate interface replacing synapsis16. Graphene layers gradually overgrown with cells act as a nanoscale implant allowing to deliver electrical stimulation reestablishing cellular communication and regulating a cells’ behaviour or intrinsic features such as contraction17.Graphene has emerged in the last decade as one of the most exciting nanomaterials whose electrical, mechanical, chemical, and structural properties can’t be obtained from either individual molecules or bulk materialsThe sandwich-structured biocomposite material was constructed from frozen human Am and covered from the stromal side with two graphene layers. The stromal side was chosen due to its ultrastructure, which is irregular and offers a suitable scaffold for graphene attachment. Therefore the graphene layer could settle freely on pleated Am surface.Am was obtained according to the well-established protocol used in ophthalmology for Am transplantation Shortly, the donors was screened to exclude the risk of transmissible infectious diseases. The Am was obtained under sterile conditions after the elective caesarian section was washed out with an antibiotic solution, and the chorion was separated manually. With the epithelial surface up, Am was spread uniformly on nitrocellulose membranes. Am was then placed in a preservative medium and stored at −80 °C.Am harvesting and experiment protocols were approved by Ethics Committee of Collegium Medicum, University of Nicolaus Copernicus . All methods were carried out in accordance with national regulations on ethics and research in EU. Am was harvested from donors above 18 years old after obtaining written permission. All donors were informed consent for study participation.18.Transferring graphene onto scaffolds required an individual approach to the transfer methods. Traditional PMMA (Polymethyl-methacrylate) approaches can’t be used since graphene’s adhesion to polymers is stronger than to extended surfaces, which Am is. As a result, graphene is removed from the substrate scaffolds during the dissolving of PMMA. To overcome this issue, we applied a modified marker-frame method Fig. 18.Figure20. Then a PDMS (Polydimethylsiloxane) frame instead of marker frame was positioned on a surface of bilayer graphene. Next, a sample was placed on the surface of an aqueous solution of ammonium persulfate (1 M) . After approximately 2 hours when Cu foil was completely etched, and bilayer graphene inside the PDMS frame floats on the surface of the solution, the sample was cleaned with a continuous and controlled flow of DI water. In the end, the bilayer graphene with a PDMS frame was carefully fished by Am and left at room temperature to dry and to improve both adhesion and contact between graphene and Am. Finally, the PDMS frame was mechanically removed from the Am, leaving the graphene layer alone on its surface.In this work, we used commercially available graphene (Graphenea Inc. USA) on 18 µm thick copper (Cu) foil. Since the marker-frame method is very demanding in terms of handling fragile graphene, at the very first step, we aimed at creating a bilayer of graphene on the copper substrate. We transferred the monolayer of graphene from the copper foil onto graphene on the copper foil by using electrochemical delamination methods21. All biocomposite samples (n = 5) included the same Am used as control (n = 5). The tested samples  were mounted into flat grips with a gauge base of 10 mm. During the test, the specimen was longitudinally stretched at a rate of 0.2 mm/s until failure. The grip travel and specimen load were continuously measured over the test procedure with a precision force transducer  and a MTS system linear variable differential transformer . The Young’s elastic modulus (MPa) was estimated based on generated Stress/strain curves.Tensile tests were conducted on a load frame of a servohydraulic material testing machine  as it was described previouslyin vitro. It was designed also for Electrical Field Stimulation (EFS) of cells seeded on biomaterial fixed in CellCrown . To our knowledge, there isn’t any commercially available testing system for this purpose. Electrical conductivity was measured using a four-sensing probe. The custom made probe utilized four 4 mm diameter electrodes printed from graphene PLA filament   spaced 2 mm apart. This gap was adjusted to graphene layer dimensions (10mmx10mm). Graphene containing filament was recently shown to be a suitable material for the fabrication of customized 3D printed electrodes22. The low-frequency (100 Hz) electrical conductivity at room-temperature was calculated for samples (n = 5) of each material. As a control, Am without a graphene layer and porcine bladder wall were utilized. The measurement setup consisted of the following devices: AFG3251 Arbitrary Function Generator , DMM4050 multimeter  and DMM4040 Osciloscope . Raspberry Pi  computer gathered the data and calculated the conductivity of each material based on its electrical resistivity  filament  Fig.  All of tity Fig. .Figure 2et al.23. Accordingly, urinary bladders from male domestic pigs were obtained from the local abattoir. All isolation and culture procedures were carried out in a laminar flow hood, using sterile techniques. The dissected smooth muscle layer of the urinary bladder was cut into small pieces and digested in collagenase II  and dispase II  solution . After digestion, the resulting suspension was filtered through 100μm nylon cell strainer  and centrifuged at 1500 × g for 5 min. Isolated cells were counted and seeded at a density of 2 × 104 cells/cm2 in SmGM-2 culture medium . Bladder smooth muscle cells were cultured up to the third passage.Isolation and culture of bladder smooth muscle cells were carried out according to the protocol of Pokrywczyńska et al.24. Accordingly, urothelial cells were isolated from porcine urinary bladders harvested from male domestic pigs during the planned economic slaughter in a local slaughterhouse. All isolation and culture procedures were carried out in a laminar flow hood, using sterile techniques. Bladder tissue pieces (1 × 1 cm) were incubated for 15 h at 4 °C in the conical tubes containing 10 mL dispase II  dissolved in the Hank’s Balanced Salt Solution (HBSS) (2.4 U/mL). After the dispase inactivation with an equal volume of DMEM/Ham’s F12 medium supplemented with FBS, the urothelial cells were carefully scraped with the blunt side of a scalpel, and the remaining tissue was discarded. Separated white conglomerates were transferred into a falcon tube and centrifuged at 1000 × g for 5 min. Isolated cells were counted using a trypan blue and seeded at a density of 4 × 104 cells/cm2 in the commercially available CnT-57 growth medium . Urothelial cells were cultured up to the third passage in standard conditions at 37 °C with 5% CO2 atmosphere and 98% humidity until reaching 75–90% confluence.For isolation and culture of urothelial cells, we used the earlier optimized protocol of Pokrywczyńska 2) and cultured until confluency was reached. The first stage of analysis included evaluation of cells morphology and growth pattern . Then multi-staining  was used to determine the viability of cultivated cells on the graphene covered surface. The number of viable cells was estimated as follows. The number of ethidium positive cells was subtracted from the number of DAPI positive cells. Labeled cells were counted with a confocal microscope  within the circular ROI (Region of Interest) (n = 10). The analysis was performed at 40x magnification with particle analysis function in ImageJ (https://github.com/imagej/imagej).Glass coverslips covered with a graphene layer (n = 20) were applied to evaluate graphene cytotoxicity to SMCs. As a control group, coverslips without graphene were used (n = 20). SMCs were seeded (20 000 cells/cm2). Subsequently adherent cells were counted within ROI (n = 5) 3 h, 6 h and 12 h after seeding.Glass coverslips with a graphene layer (n = 5) were applied to evaluate SMCs adherence to surface covered with graphene. As a control group, coverslips without graphene were used (n = 5). SMCs were seeded  derived from SMCs seeded biocomposite grafts (n = 5) were fixed in 10% (v/v) neutral (pH = 7) buffered formalin and embedded in paraffin. Cross-sections of the entire area covered with graphene were prepared, and histological analysis using H&E staining was performed. Additional immunohistochemical staining using anti-SMA antibodies  and anti-smoothelin antibodies  was conducted to identify SMA and smoothelin cells and to distinguish them from the Am matrix. Stanning for smoothelin was conducted to verify whether in vitro cultured SMCs preserved their mature contractile phenotype. Digital images of anti-SMA and anti-smoothelin stained specimens were used for quantitative evaluation of SMC content. Immunohistochemistry Image Analysis Toolbox plugin (https://imagej.nih.gov/ij/plugins/) for ImageJ was applied to calculate SMA stained surface using generated histograms. The evaluation, according to this protocol, was done in square ROI (n = 10) for each specimen.After six days of in vitro culture, multi-staining  was used to determine viability and arrangement of UCs cultivated on biocomposite and Am surface. Labelled cells identified as alive were counted with a confocal microscope  by creating a square region-of-interest (ROI) (n = 10) at 40× magnification.After 7 days of in vitro cultured SMC was conducted using an electrode printed from conductive graphene PLA filament  . The measurement of the temperature was conducted 15 minutes after removing stimulator form cultivation incubator at room temperature. Following stimulation period cells were cultured an additional 48 hours. The multi-staining  was used to determine the impact of EFS on cell number and arrangement. Labelled cells identified as alive were counted with a confocal microscope  by creating a square (ROI) (n = 10) at 40× magnification. The orientation of cultivated SMC was established by a square ROI (80 × 80 pixels) (n = 10) analysis using ImageJ OrientationJ  plugin. SMC bodies were manually outlined on DAPI/Calceine threshold images by fitting an ellipse to the selected region. The average cell orientation was determined by averaging all the values of deflection angles obtained from the ROI (n = 10).Electrical stimulation of SA) Fig.  The gap We have designed an experimental setup to elicit false contractions from the cultivated muscle layer and record the increase of the pressure generated by the contracted graft Fig. . Each anCalculated results were reported as means with standard deviations. Statistical difference was evaluated using a one-way analysis of variance (ANOVA) with Tukey’s post hoc comparison at a significance level of 0.05.−1 and the 2D band at ~2688 cm−1, which confirms the presence of a graphene structure27. Evaluation of Young’s elastic modulus (E) indicated that either graphene layer itself or method of graphene transfer didn’t influence mechanical characteristic of Am which preserved its native elasticity  and reference scaffold substrate (red line) are present Fig. . For theity Fig. . The manDesign printed stimulator allowed to precisely lower the electrodes to provide comprehensive and stable contact points. The calculated electrical conductivity of the graphene-based biocomposite, Am alone, and porcine urinary bladder was demonstrated in Fig. . The graDue to the presence of the graphene layer, we didn’t notice an abnormal morphology of cultured SMCs. The cells were characterized by normal spindle shape, and the confluence of the cell populations was indistinguishable between groups. The graphene layer didn’t exert any cytotoxic effect or influence the SMCs adhesive properties. SMSs formed typical homogenous monolayer with single clusters. The number of cells cultivated on the graphene layer was comparable between groups, and the graphene layer didn’t decrease cell survival. The estimated cell number between groups is shown in Fig. in vitro cultivation. Graphene, as a hydrophobic carbon material didn’t impair the cells adhesive capability. Anti-SMA and anti-smoothelin staining were more intense on the biocomposite surface than Am alone . There is a need to analyze behavior of graphene layer in context of its elasticity and resistance for repeating load and unload cycles typical for urinary tract environment.Since the development of graphene, this carbon material has been evaluated for application in tissue engineering. Graphene is used to design multifunctional biomaterials that can provide physical, electrical, and structural signalling to cells and tissues11. An aqueous solution of ammonium persulfate is widely used for nanomaterial preparation. This agent, due to excellent solubility in water is easily washed away from the graphene layer. The concept of a stable graphene layer transfer is known in the electronics industry, but hitherto only metallic or polymeric materials acted as substrates33. This study introduced a new application for this technology in the field of biomaterials. The demonstrated method may be adopted to cover different biomaterials with graphene to fabricate conductive biohybrid materials that may find application in cardiologic or neurologic tissue engineering. The advantage of applied methodology for graphene transfer is the potential possibility to preserve intact Am bioactive content and mechanical properties. Nevertheless this assumption needs to be verified in further studies.Am was extensively evaluated for urological tissue engineering, also in clinical settings35. Bladder detrusor has a unique histological structure with a syncytial ultrastructure divided into autonomous functional units connected by distributed innervation and a network of intestinal cells36. In contrast to striated muscle tissue, bladder’s smooth muscle syncytium creates a different environment for automatic propagation of action potentials37. From the electrophysiological point of view, local electrical stimulation tends to “scatter” in syncytium evoking action potentials with different inefficient shapes and sizes without a contractile response. In this situation, the external electrical pulse aimed to trigger an action potential must be simultaneously delivered at multiple points. Due to the excellent electrical properties of graphene, it offers the technological possibility to address this challenge by establishing an efficient biomaterial-cellular interface mediated coordinated response. In this context, graphene may become a milestone technical solution allowing to replace neuronal networks in artificial organs.Graphene-based biomaterials were introduced as scaffolds supporting muscle cell proliferation. Most of them, however, were indented to promote myogenic differentiation and proliferation of striated muscle or cardiocytes38. We believe that the direct contact and exposure of SMCs to the electric charge was crucial to evoke an active contraction pattern. Moreover, graphene’s natural high flexibility prevented cells from detachment during construct manipulation and contraction itself. This graphene feature is particularly crucial for biomaterials indented to replace hollow organs that change volume during normal functioning such as the urinary bladder. The registered contractile activity of SMCs seeded on biocomposite was the result of graphene-supported stimulation propagation and a proper cell arrangement which enabled to generate stable force tensioning the Am. In comparison to cardiomyocytes electrophysiology of detrusor, SMCs is less known in vitro. Thus the applied stimulation parameters were chosen based on a few published studies concerning intact detrusor stimulation in vitro41. Parameters of electrical stimulation were chosen assuming that activation threshold of tissue engineered layer of SMCs should be similar to normal muscular layer of bladder wall.The graphene layer as a nanomaterial interacts with single cells promoting tight adhesion between cell membranes and the interfacing biomaterialin vitro, including the lack of a reference protocol. Kobayashi et al. determined that intestinal SMS seeded electrospun poly(3-caprolactone) scaffolds exhibited rhythmic contractions resulting in biomaterial contraction42. Krueger et al. reported the ability of muscle cells, forming myotubes seeded on graphene foam to mediate consolidated micromovements43. The designed, printed chamber, mimicking the bladder environment might become useful to evaluate the contraction ability of tissue-engineered grafts planned for reconstructive urology. We hypothesize that the fabricated chamber evaluating the tissue-engineered construct contractility based on pressure changes might be more sensitive in a tissue-engineered scenario than in stripe testing. It is challenging to obtain an SMCs seeded biomaterial with the ability to generate a singular vector contraction force recognized by a force transducer connected at both ends.There is a shortage of literature demonstrating contraction of tissue-engineered grafts 44. The exact mechanism responsible for this phenomenon is unknown. Mainly, it is unclear whether graphene acts as a passive molecular pathway for proliferating cells or instead actively interacting with adhesive membrane molecules. Graphene incorporated with a collagen scaffold likely created micro-topographic structures that were preferably recognized by migrating neurons or muscle cells45. Moreover, being over one hundred-fold smaller than cells, graphene shouldn’t negatively affect the cytoarchitecture of the regenerating tissue contributing to the functionality of the reconstructed bladder wall.The role of modern biomaterials must include the ability to influence the cell arrangement to restore proper tissue architecture. The graphene layer passively organized the distribution of seeded SMCs and UCs. In the case of SMCs, it also directed cell orientation during EFS. From the beginning of research on the biotechnological application, the graphene ability to order cell arrangement is underlined46. Hydrophobic character of graphene may also reduce contact of regenerating tissue without developed urothelial barrier with urine. Urine has recognized cytotoxic activity which may impair regeneration47.From the perspective application in urology, a graphene-based biomaterial would also have additional advantages particularly important for clinical practice. The known antibacterial activity of graphene should prevent infection and facilitate healing of urinary tract wall after future reconstructive proceduresin vitro. Further research are needed to evaluate behavior of this scaffold on in vivo models. Utilization of graphene for biomaterial design might become the first step to obtain biocybernetics platform aimed to control and regulate the function of tissue engineered bladder.The developed graphene based biocomposite successfully managed to deliver and to propagate external electrical stimulation to cultivated smooth muscle cells. The combination of conductive graphene and Am supporting cell growth resulted in creation of tissue engineered grafts capable of induced contraction