IR spectroscopy of [benzene-(H2S)n]+ (n = 1–4) elucidates the change of the positive charge accommodation motif from the S∴π hemibond to the S∴S hemibond. 2S)n]+ (n = 1–4) radical cation clusters. In n = 1, the CH stretch and SH stretch bands of the benzene and H2S moieties, respectively, are clearly different from those of the neutral molecules but similar to those of the ionic species. These vibrational features show that the positive charge is delocalized over the cluster due to the S∴π hemibond formation. In n = 2–4, the S∴S hemibond and S–π–S multicenter hemibond  can compete with the S∴π hemibond. The observed vibrational features clearly indicate that the S∴S hemibond formation is superior to the S∴π hemibond and S–π–S multicenter hemibond. Calculations of several dispersion-corrected density functionals are compared with the observations. While all the tested functionals qualitatively catch the feature of the S∴π hemibond, the energy order among the isomers of the different hemibond motifs strongly depends on the functionals. These results demonstrate that the [benzene-(H2S)n]+ clusters can be a benchmark of density functionals to evaluate the sulfur hemibonds.The S∴π hemibond  is an attractive interaction between a sulfur atom and π electrons. The S∴π hemibond is of essential importance in understanding chemistry of sulfur radical cations, and its roles in biochemistry have recently attracted much interest. In the present study, we observe the S∴π hemibond in the simplest model system in the gas phase. Infrared spectroscopy is applied to the [benzene-(H The S∴π hemibond has attracted great interest because it plays crucial roles in chemistry and biochemistry of sulfur radical cations.e.g., methionine–phenyl, facilitates oxidation through the stabilization by the S∴π hemibond formation.9The S∴π hemibond  is an attractive interaction between a singly occupied lone pair orbital of sulfur and a doubly occupied π orbital, or 2S)n]+, (n = 1–4), are studied by infrared (IR) spectroscopy in the SH and CH stretch regions. Since IR spectroscopy is sensitive to molecular structures and intermolecular interactions, IR spectroscopy of S∴π hemibonded systems would provide us rich information on the nature of the S∴π hemibond, which is complementary to the previous electronic and photoelectron studies.n = 1 cluster can be the simplest prototype of the S∴π hemibond in the gas phase. The observed vibrational features clearly reveal the S∴π hemibond formation in the radical cation cluster. In the n = 2–4 clusters, other hemibond motifs, the S∴S hemibond and S–π–S multicenter hemibond  can compete with the S∴π hemibond. The S∴S hemibond formation in (H2S)n+ has been observed by the transient electronic absorption in aqueous solution,n = 2–4 clusters show that S∴S hemibond formation among H2S molecules is superior to the S∴π hemibond and S–π–S multicenter hemibond. The observed spectra are also compared with the spectral simulations by density functional theory (DFT) calculations. As has been pointed out, the validity of DFT calculations of hemibonds strongly depends on functionals.2S)n]+ clusters can be a benchmark to evaluate the performance of functionals on the simulation of the hemibond motifs.For characterization of an intermolecular interaction, gas phase spectroscopy of its model clusters can provide the most reliable data, which can be also compared directly with high precision quantum chemical computations.2S)1]+ to test the existence of its stable isomers. One method is resonance-enhanced multiphoton ionization (REMPI) of the neutral Bz-(H2S)1 cluster under the collision free condition. The gaseous mixture of He/H2S/Bz was expanded to a vacuum chamber, and the resultant supersonic jet was skimmed to form a molecular beam. The [Bz-(H2S)1]+ radical cation was prepared by one-color REMPI of neutral Bz-(H2S)1via its S1–S0 610 band.2S)1 have revealed that H2S locates on the C6 axis of the aromatic ring in S0,2S molecule should locate on the phenyl ring. The produced ions were detected by a time-of-flight mass spectrometer. An IR spectrum of the [Bz-(H2S)1]+ radical cation was measured by IR dissociation spectroscopy. The IR light pulse was introduced 50 ns after the ionization light pulse, and the IR light frequency was scanned. The depletion of the [Bz-(H2S)1]+ signal due to the vibrational predissociation was detected as a measure of the IR absorption.Two different preparation methods were applied for [Bz-(H2S)1]+ cluster ion was generated by following collisions with H2S in the supersonic jet expansion. In this “pick-up” method, the most stable structure tends to be produced.n = 2–4 cluster ions were also produced by the pick-up method and their IR spectra were measured by photodissociation spectroscopy using the tandem quadrupole mass spectrometer. In all the IR spectral measurements of n = 1–4 by using the tandem quadrupole mass spectrometer, the [Bz-(H2S)n–1]+ fragment cation was monitored. No signal was detected in the (H2S)n+ fragment channel.Another method is ionization of bare Bz molecules in the collisional region of the jet expansion. Bare Bz cations were first produced by REMPI of bare neutral Bz, and the +, however, we failed in MP2 calculations because of the significant spin contamination. Therefore, we employed DFT to calculate energy-optimized structures and their IR spectra. The four dispersion-corrected functionals, B3LYP-D3, M06-2X, M06-L, and ωB97X-D, were used with the 6-311G+ and aug-cc-pVDZ basis sets. Energy-optimized structure search and harmonic vibrational simulations were performed by the Gaussian 09 and 16 program suites.In our previous studies on the sulfur-containing charged clusters, (H2S)1]+ radical cation prepared through REMPI of neutral Bz-(H2S)1 in the molecular beam. To qualitatively interpret the spectrum of [Bz-(H2S)1]+, IR spectra of bare neutral Bz+-Ar2S2S)4+2S)+, this Fermi triad disappears and only a single weak band is found at 3084 cm–1. This feature rather resembles that in Bz+-Ar at 3095 cm–1, which is supposed to be essentially identical with the bare Bz cation.2S)1]+ should be charged, as easily expected by the lower ionization energy of Bz (9.24 eV) than H2S (10.46 eV).–1 and this band is largely red-shifted from those of neutral H2S.2S and Bz. This is because the aromatic ring is positively charged, as shown by its CH stretch band, and the aromatic ring rather repels the proton (hydrogen) of H2S.2S directly solvates a charged site, charge transfer occurs more or less, and it lowers the SH stretch frequencies because of the partial reduction of the electron density in the SH bonds. The IR spectra of (H2S)n+ (n = 3–6) and H+(H2S)n+ (n = 3–9)2S molecule in the first solvation shell of a charged site are seen only in the region higher than 2585 cm–1. The 2593 cm–1 band of (H2S)4+ reproduced in 2S molecules in the first solvation shell to the ion core. The observed SH band frequency of [Bz-(H2S)1]+ is too low to be assigned to neutral H2S, but it is very close to that of the free SH stretch (2565 cm–1) of the ion core moiety in (H2S)4+.2S)4+ has the hemibonded ion core, (H2S∴SH2)+, in which the positive charge is equally shared by the two H2S molecules.2S)1]+ is largely shared by the H2S moiety, i.e., an S∴π hemibond is formed in the cation. The CH stretch frequency in (Bz)2+, in which the two Bz molecules are π∴π hemibonded (in the charge resonance state),–1 from the dimer ion core component in the IR spectra of (Bz)n+ (n ≥ 3).2S)1]+ is actually located in between those of bare Bz+ and (Bz)2+, and this is consistent with the S∴π “hetero” hemibond, in which the excess charge cannot be equally shared by the two different molecules.2S)1]+ formed by the two different ion sources. 2S)1]+.2S)1]+. To confirm the above qualitative discussion on the observed IR spectrum by the comparison with the related species, dispersion-corrected DFT calculations were performed with four functionals  and two basis sets  and aug-cc-pVDZ). Both the basis sets provided similar results for each functionals. Then, in the following, we focus on the results with the 6-311G+ basis set, and those of the aug-cc-pVDZ basis set are summarized in the ESI.This S∴π hemibond formation would be a unique interpretation to reasonably explain the observed IR spectrum of [Bz-(H2S)1]+, and all of them finally converged to a unique on-top structure. This is consistent with the missing of apparent isomers in the observed spectra. The stable structure (1-1) at the ωB97X-D/6-311G+ level is shown in the left column of 2S have the 3p character and are almost perpendicular to the SH bonds, the optimized structure suggests large overlap between the non-bonding orbital of H2S and the π orbital of Bz. The spin density of this structure is also shown in the right column of 2S moieties. The ionization energy of H2S is, however, about 1.2 eV higher than Bz, and the charge distribution cannot be equivalent in the two moieties. The natural charges on the H2S and Bz moieties are calculated to be 0.438 and 0.562, respectively.Several initial on-top structures as well as in-plane (CH–S hydrogen-bonded type) structures were tried in the energy optimization of [Bz-(H2S)1]+ by the four functionals with the 6-311G+ basis set are shown in –1, and were also normalized to have the same peak maximum of the SH band. Agreement between the observed and simulated spectra is not perfect; the position and relative peak intensity of the CH stretch band show small differences from the observed ones. However, all the simulated spectra reproduce well the gross features of the SH and CH band positions and their relative intensities of the observed spectra. These simulations strongly support the S∴π hemibond formation in [Bz-(H2S)1]+. Moreover, these simulations demonstrate that all the four functionals are useful to catch the physical essence of the S∴π hemibond.The harmonic vibrational spectra of the S∴π hemibonded structure of [Bz-(H2S)1]+ is quite different from that of the water analogue cluster, [Bz-(H2O)1]+.2O)1]+, stable on-top structures attributed to the O∴π hemibond have been predicted in the theoretical computations. However, the in-plane structure formed by the CH–O hydrogen bonds is much more stable, and only this structure has been experimentally identified. The finding of the stable in-plane structure in [Bz-(H2O)1]+, which is missing in [Bz-(H2S)1]+, can be attributed the fact that the difference of the ionization energy between Bz and H2O (3.4 eV) is much larger than that between Bz and H2S (1.2 eV).2O)1]+ might be essentially regarded as a charge-dipole complex), and the potential minimum in the aromatic ring plane can independently exist. In the in-plane structure of [Bz-(H2O)1]+, the CH stretch band intensity is remarkably enhanced by the CH–O hydrogen bond, and the CH band appears as strong as the OH stretch bands.2S)1]+, in which the SH band is much stronger than the CH band, and also supports the S∴π hemibonded structure of [Bz-(H2S)1]+.It is worth to note that the present result on [Bz-(H2S)n]+ (n = 1–4), which are produced by the pick-up type ion source. In the following, the cluster size is simply denoted only with n, the number of H2S molecules in [Bz-(H2S)n]+. In the spectra of the n > 1 clusters, several new features are seen, in addition to the free SH of the ion core (∼2560 cm–1) and CH stretches of Bz (3000–3100 cm–1). By the comparison with the previously reported IR spectra of (H2S)n+ and H+(H2S)n,–1 are attributed to H-bonded SH stretches of the ion core .–1) at the high frequency side of the free SH of the ion core is assigned to free SH stretches of the neutral H2S moiety.n > 1 clusters, multiple H2S molecules enable several different hemibond motifs. The observed IR spectral features provide us rich information on the competition among the hemibond motifs.n = 2 cluster at the ωB97X-D/6-311G+ level, and these stable structures and their spin density plots are shown in 2-1, Bz is sandwiched by two H2S molecules. The spin density plot shows that the charge is delocalized to all the three molecules, and this means that the S–π–S multicenter hemibond (3c-5e bond) is formed. Structure 2-2 is a variation of the multicenter hemibond; two S∴π hemibonds are formed on the carbon atoms in the diagonal position, and the charge is delocalized to all the molecules. Structure 2-3 holds the single S∴π hemibond. The second H2S molecule is essentially neutral and is H-bonded to the hemibonded H2S (ion core). In structure 2-4, the two H2S molecules form an S∴S hemibonded ion core, (H2S∴SH2)+, and the ion core is solvated by the neutral Bz molecule. These stable structures demonstrate that three different hemibond motifs, S–π–S, S∴π, and S∴S, can compete in the present system.Four different types of hemibond motifs are found in the stable structure search of the The relative energy of each isomer structure is summarized in n = 2 cluster at the ωB97X-D/6-311G+ and B3LYP-D3/6-311G+ levels are shown in 2S moiety, and green: CH stretch. Note that each arrow indicates a peak of an envelope in which contribution of multiple vibrational modes can be involved. Bands without an arrow are attributed to H-bonded SH stretch of the ion core, and they appear only below 2400 cm–1. The free SH bands of the ion core and neutral H2S moiety appear in 2540–2600 cm–1, and the band patterns (symmetric/antisymmetric SH stretches or dangling SH stretch) depend on the structures. The splitting between the symmetric and antisymmetric SH stretch bands and their intensity distributions depend also on the functionals.The simulated IR spectra of the four isomers of the n = 2 cluster shows two H-bonded SH bands (2235 and 2340 cm–1) of the ion core (charged) moiety. To reproduce these two bands in the low frequency region, coexistence of structures 2-3 and 2-4, each of which shows a single strong H-bonded SH band of the ion core, is clearly requested. In both the M06-2X and M06-L calculations, however, the energy of structure 2-3 relative to structure 2-4 is much higher, and its coexistence is practically excluded. Therefore, the M06-2X and M06-L results conflict with the observation, and these two functionals are omitted in the following discussion. The remaining two functionals, ωB97X-D and B3LYP-D3, provide the totally different energy evaluations of the hemibond motifs. While structures 2-4 and 2-3 are the most stable isomers in ωB97X-D, structure 2-1 is most stable and structure 2-4 is a rather high energy isomer in B3LYP-D3. Structure 2-1 shows only the free SH stretch band of the ion core, which can correspond to the band at 2564 cm–1 in the observed spectrum. But this observed band can be also attributed to structures 2-3 and 2-4. The correct evaluation of the relative intensities of the free SH stretch and H-bonded SH stretch bands is practically difficult from the observed spectrum because of the large intensity difference between these bands. Therefore, we cannot estimate the contribution of structure 2-1 (and/or 2-2) in the observed spectrum of n = 2. The matching of the spectral simulation with the observed spectrum is slightly better in B3LYP-D3 than ωB97X-D. However, the minor difference in the spectral simulations does not affect the assignments of the observed bands, and this cannot be a critical issue in the present case. Further examination of the reliability of the functionals is difficult for n = 2. Thus, we examine the spectra of n = 3 and 4.The observed IR spectra of the n = 3 and 4 shown in –1, which is highlighted by the dashed line. This band becomes weaker with increasing cluster size, and finally disappears at n = 4, while the free SH stretch band of the neutral moiety is still seen at 2599 cm–1. This weakening of the free SH band of the ion core reflects the progress of its solvation (H-bond formation) by H2S. The ion core of structure 2-1 (2-2) is the whole cluster. Both the two H2S molecules are not H-bonded at all, and they have totally 4 free SH bonds. In structure 2-3, the H2S molecule in the ion core (H2S bound by the S∴π hemibond) is H-bonded to a neutral H2S molecule. Therefore, the ion core has only 1 free SH bond. In structure 2-4, the ion core is the S∴S hemibonded (H2S)2+ dimer moiety. This ion core is bound to the Bz moiety with the two SH/π H-bonds, and the core has totally 2 free SH bonds. Therefore, each ion core of structures 2-1, 2-3, and 2-4 has 4, 1, and 2 free SH bonds, respectively . Therefore, the free SH stretch band of the ion core should disappear at n  = 6, 3, and 4 for structure 2-1, 2-3, and 2-4 type ion cores, respectively, with the completion of the first solvation shell of the ion core. The observed disappearance of the free SH stretch band of the ion core at n = 4 is a clear indication of the structure 2-4 type (S∴S hemibonded) ion core in n = 4.In the observed spectra of 2-4 type ion core is also evidenced by the spectral change in the CH stretch region. The CH stretch region of the observed spectra of the n = 1–4 clusters is reproduced in n = 1 shows the single and broadened band, which is quite similar to the CH stretch band of bare Bz+ (Bz+-Ar),n = 3 cluster shows the clear Fermi triad structure, which indicates that the Bz moiety is essentially neutral.n = 2), only structure 2-4 has the neutral Bz moiety. Therefore the spectra of the CH stretch region demonstrate that the major isomer of n = 3 has the structure 2-4 type ion core. Unfortunately, the CH stretch band of the n = 4 cluster is hardly seen because of its poor signal to noise ratio.The preference of the structure n = 3 and 4 were performed by the ωB97X-D and B3LYP-D3 functionals with the 6-311G+ basis set. As for n = 3, the energy optimized structures and their relative energies are summarized in 3-1, 3-2, 3-3, and 3-4 shown in 2-1, 2-2, 2-3, and 2-4, respectively, and one more H2S molecule solvates the ion core moiety by an H-bond. In both the computational levels, structure 3-4 of the S∴S hemibonded motif is the most stable isomer. However, other isomers are much higher in energy in ωB97X-D, while structures 3-1, 3-3, and 3-4 are almost degenerated in B3LYP-D3. The vibrational simulations based on these structures are summarized in ESI.–1) in the observed spectrum, dominant population of 3-3, which lacks free SH in the ion core, is easily excluded. As the case of n = 2, it is practically difficult to uniquely identify the contribution of isomers by the comparison of the SH stretch region. As shown in 3-4, in which the Bz moiety is not involved in the ion core . Therefore, the ωB97X-D computation well reproduces the observed spectral feature, and the energetics evaluated by B3LYP-D3, which predicts coexistence of structures 3-1, 3-3, and 3-4, is not consistent with the observation.Computations of stable structures of n = 4 and their relative energies are summarized in 4-1, 4-2, and 4-4 are based on structures 3-1 (2-1), 3-2 (2-2), and 3-4 (2-4), respectively. Both structures 4-3a and 4-3b are based on 3-3 (2-3). In all these structures, the ion core moiety is solvated by one more H2S molecule than n = 3. In n = 4, both the calculation levels show that structure 4-4 which has the S∴S hemibonded ion core is the most stable structure. Other isomers are much higher in energy, and the exclusive population of structure 4-4 is predicted. The vibrational simulations by these structures are also summarized in ESI.4-3 and 4-4 have no free SH in the ion core, and well reproduce the missing of the free SH band of the ion core in the observed spectrum. As was already pointed out in the above qualitative discussion, the missing of the free SH band of the ion core occurs at n = 4, not at n = 3. Moreover, an H-bonded SH stretch band of the neutral moiety is expected in 2500–2600 cm–1 region for structure 4-3 (both a and b), but such a band is missing in the observed spectrum. All these observed features indicate the dominance of structure 4-4 in n = 4, and this supports the computational prediction.The energy optimized structures of n = 2 shows the coexistence of structures 2-3 (S∴π hemibonded) and 2-4 (S∴S hemibonded), and those of n = 3 and 4 demonstrate the clear preference of the S∴S hemibonded type ion core in these sizes. These observations agree well with the energy evaluation of the ωB97X-D functional, but conflict with those of other functionals. Therefore, it is concluded that ωB97X-D is the best performance functional among the present four functionals to evaluate the hemibond motifs. Moreover, the ωB97X-D calculations predict that the S–π–S multicenter hemibond is much less stable than the S∴π and S∴S hemibonds. This less preference of the multicenter hemibond is also supported by the present experimental spectra of n = 3 and 4.The observed IR spectrum of 2S)n]+ (n = 1–4), were studied by IR spectroscopy combined with the DFT computations. The IR spectrum of n = 1 clearly demonstrated the formation of the S∴π hemibond in this simplest model system. In n = 2–4, the S–π–S multicenter hemibond and S∴S hemibond can compete with the S∴π hemibond. The IR spectrum of n = 2 showed the coexistence of the S∴π and S∴S hemibond motifs. The spectral features in n = 3 and 4 indicated the S∴S hemibond motif is superior to other hemibond motifs. The relative energy evaluations of these hemibond motifs strongly depend on the DFT functionals, and the ωB97X-D functional showed the best performance to reproduce the observed trend. It should be noted that the charge accommodation motifs in [Bz-(H2S)n]+ (n = 1–4) can be uniquely assigned by their spectral features essentially without help of theoretical computations. Therefore, these [Bz-(H2S)n]+ clusters can be a benchmark system to evaluate performance of DFT functionals to handle sulfur hemibonds. Moreover, they would be highly helpful to explore the nature of the S∴π hemibond by both more extensive experimental and theoretical approaches, and obtained knowledge will be the basis to discuss roles of the S∴π hemibond in biological functions.The S∴π hemibond formation and its competition with other sulfur hemibond motifs in the model clusters, [Bz-(HThere are no conflicts to declare.Supplementary informationClick here for additional data file.


Fused filament fabrication (FFF) is a promising additive manufacturing (AM) technology due to its ability to build thermoplastics parts with advantages in the design and optimization of models with complex geometries, great design flexibility, recyclability and low material waste. This technique has been extensively used for the manufacturing of conceptual prototypes rather than functional components due to the limited mechanical properties of pure thermoplastics parts. In order to improve the mechanical performance of 3D printed parts based on polymeric materials, reinforcements including nanoparticles, short or continuous fibers and other additives have been adopted. The addition of graphene nanoplatelets (GNPs) to plastic and polymers is currently under investigation as a promising method to improve their working conditions due to the good mechanical, electrical and thermal performance exhibited by graphene. Although research shows particularly promising improvement in thermal and electrical conductivities of graphene-based nanocomposites, the aim of this study is to evaluate the effect of graphene nanoplatelet reinforcement on the mechanical properties, dimensional accuracy and surface texture of 3D printed polylactic acid (PLA) structures manufactured by a desktop 3D printer. The effect of build orientation was also analyzed. Scanning Electron Microscope (SEM) images of failure samples were evaluated to determine the effects of process parameters on failure modes. It was observed that PLA-Graphene composite samples showed, in general terms, the best performance in terms of tensile and flexural stress, particularly in the case of upright orientation . In addition, PLA-Graphene composite samples showed the highest interlaminar shear strength (about 1.2 times higher than PLA and PLA 3D850 samples). However, the addition of GNPs tended to reduce the impact strength of the PLA-Graphene composite samples (PLA and PLA 3D850 samples exhibited an impact strength about 1.2–1.3 times higher than PLA-Graphene composites). Furthermore, the addition of graphene nanoplatelets did not affect, in general terms, the dimensional accuracy of the PLA-Graphene composite specimens. In addition, PLA-Graphene composite samples showed, in overall terms, the best performance in terms of surface texture, particularly when parts were printed in flat and on-edge orientations. The promising results in the present study prove the feasibility of 3D printed PLA-graphene composites for potential use in different applications such as biomedical engineering. Additive manufacturing (AM) is one of the most promising areas in the manufacturing of components from prototypes to functional structures with complex geometries and is revolutionizing different important industrial areas such as in aerospace, automotive, semiconductor or biomedical applications ,6,7,8,9.The interest in FFF abilities has expanded to include functional finished parts in addition to rapid prototyping. Moreover, the cost of manufacturing small series or unique parts can be significantly reduced . This ha3D printing of polymer composites with enhanced mechanical properties solves the previous limitations by combining the matrix and reinforcements to achieve a system with more useful structural or functional properties non-attainable by any of the constituent alone ,30. IncoThe allure of the recent introduction of nanotechnology into this innovative field is due to the remarkable improvements and diversifications in properties of the resulting 3D printed materials, exhibiting optimized properties and multifunctionality. In particular, there is increasing interest in the development of high-performance composites suitable for 3D printing, achieved via the introduction of nanomaterials with unique properties such as nanotubes and graphene and its derivatives in the polymer matrix. Graphene’s excellent mechanical, electrical and thermal properties make it an attractive candidate for the reinforcement of several polymers . GraphenIn this study, commercially available polylactic acid (PLA), an enhanced PLA-based polymer (PLA 3D850) and graphene nanoplatelet reinforced PLA composite (PLA-Graphene) filaments were used to manufacture different samples by the FFF technique using a low-cost desktop 3D printer. The mechanical properties, in terms of tensile and three-point bending performance, are evaluated. In addition, the interlaminar bonding and impact performance of the 3D printed samples are also studied. The effect of the graphene reinforcement and build orientation are analyzed. A comparison of the mechanical performance, dimensional accuracy and surface texture between virgin PLA and reinforced PLA samples is also conducted. Finally, SEM images of failure samples are evaluated to determine the effects of the process parameters on failure modes.The rest of the paper is organized as follows. First, the experimental methodology carried out in this study is briefly summarized with particular emphasis on specimen preparation, the 3D printing process and the experimental set-up. Thereafter, the key results of the investigation are summarized, and the effects of the different process parameters on the mechanical performance, dimensional accuracy and texture are highlighted. Finally, conclusions and extensions of this work are outlined.® PLA natural [® PLA 3D850 natural [® PLA (PLA-graphene) manufactured by Haydale Ltd.  [® functionalized graphene nanoplatelets (GNPs) of a planar size between 0.3–5 μm in order to improve dispersion and bonding within the PLA polymer. It is expected to improve thermal stability, print quality, the first layer and z-axis adhesion. The goal of this study is to analyze the mechanical performance, dimensional accuracy and texture of PLA-graphene composite samples. Three different commercially available PLA-based filaments, with a diameter of 1.75 mm, have been analyzed: SMARTFIL natural , a modif natural , both fiire, UK) . PLA 3D8The basic mechanical properties of PLA-based materials used in this work and typical ranges of mechanical properties for PLA and ABS materials manufactured by FFF technology provided by the manufacturers are presented in PLA-based samples were manufactured using a WitBox desktop 3D printer developed by BQ . WitBox There are no standard test methods for the mechanical characterization of parts manufactured using FFF. In this study, the recommendations of the ASTM standards D638 , D790 5, D6110-1The mechanical, dimensional and surface finish properties of parts fabricated using FFF technology depend on the selection of process parameters ,13. In tThere is a broad spectrum of infill patterns, making it difficult to analyze the influence of raster patterns. In this study, solid samples filled with a perimeter raster were analyzed, which is where shell thickness was selected long enough to fill the sample with a raster angle of 0° d. In othEach sample set consisted of five specimens for each orientation and material, with a total of 150 specimens for mechanical testing  and 45 specimens for dimensional and surface roughness characterization. Average values of the mechanical, dimensional and surface roughness tests were taken as the results. The manufactured specimens were stored in a dry box in order to minimize moisture absorption, which adversely affects the mechanical performance of PLA. Since the physical properties of many materials  can vary depending on ambient temperature, tests were carried out according to the standards for room temperature.A 50 kN universal electro-mechanical testing machine with a 5 kN load cell was used for the uniaxial tensile, 3-point bending and ILSS tests at a fixed loading rate of 2 mm/min, for both the tensile and 3-point bending tests, and 1 mm/min for short beam shear test (SBS) to obtain the ILSS strength. The selection of this displacement rate was in agreement with the displacement rate used in other studies ,13,39 an= 2.0 + 10L/1000 µm, where L is the machine axis length in mm. In addition, surface texture was measured using a 3D Surface Profiling System Talysurf CLI 1000 with a range of 100 mm in the three axes . The Talysurf CLI 1000 system uses an inductive contact gauge with a 2.5 mm Z-axis range and a resolution of 40 nm. In addition, surface texture was measured by sampling a 10 x10 mm2 area in a flat position on the surface center point of the workpiece upper face, taking 334 profiles separated 24 µm. The surface roughness parameters analyzed were the Arithmetic Mean Height (Sa) and the Maximum height (Sz) [Root Mean Square Flatness Deviation (Fltq) with Least Squares Reference Plane (LSPL) [For dimensional inspection, the optical measurement used a vision system Tesa-Visio 200 with a motorized zoom and CCD color camera. The axis ranges are X-200 mm, Y-100 mm, Z-150 mm, with a resolution of 0.001 mm; repeatability is defined by Ex, Ey ght (Sz)  and for e (LSPL) .t, σf) and stiffness  of the 3D printed PLA-based samples are tabulated in C) and the maximum interlaminar shear strength (τILSS) are tabulated in Average and standard deviation of the test results of the maximum tensile and flexural strength  as a function of the PLA material and build orientation. It can be observed that On-edge and Flat orientations showed higher values of impact strength than upright orientation. In the case of Flat samples, impact loading was parallel to the adjacent layers and they withstood most of the applied load. On the other hand, in the case of On-edge samples, impact loading was perpendicular to the individual layers, and they withstood most of the applied load. In addition, Flat samples exhibited the highest value of impact strength for the three PLA-based materials. These results were in line with those reported in references [ferences ,65 for uferences .Ec, of the PLA-Graphene specimens with PLA and PLA 3D850 specimens, it is observed that the impact strength, Ec, of PLA and PLA 3D850 samples was about 1.2–1.3 times higher than the maximum impact strength of PLA-Graphene composite samples. Hence, the addition of GNPs tended to reduce the impact strength of the PLA composite samples. This can be explained by the brittle behavior exhibited by the PLA-Graphene composite samples in On-edge and Flat orientations. On the other hand, in the case of Upright oriented samples, PLA-Graphene composite samples showed similar or even higher ductility and mechanical performance compared to PLA and PLA 3D850 samples. This trend indicates an enhanced interlayer adhesion and it is in agreement with the findings of the previous section.Comparing impact strength, The bonding performance of the different layers is a main factor for the mechanical strength of 3D printed thermoplastic parts. In addition, Finally, SEM examination of the cross-sectional ILSS samples was performed to obtain information on the interlayer performance. X-axis, Y-axis and Z-axis, respectively, showing the mean, the maximum and the minimum values of the three materials and build orientations analyzed in this study. The results of three samples of each set are shown. X-axis and Y-axis are related to the dimensional accuracy of deposited filaments in the same layer. On the other hand, Z-axis is associated with the dimensional accuracy of deposited layers. The results showed that the greatest differences in dimensional accuracy are linked with the build orientation. In the case of the X-axis, Flat and On-edge orientations depicted higher dimensional deviation than Upright orientation. More specifically, flat and on-edge PLA 3D samples showed a mean deviation of 288 ± 13 µm and 304 ± 16 µm, respectively, while upright PLA samples depicted a mean deviation of 78 ± 48 µm. The reason was that Upright orientation was affected by layer accumulation and a lower dimensional deviation was expected. In the case of the Y-axis, it depicted the highest mean dimensional deviation of all test with a maximum mean deviation of 407 ± 9 µm for Flat PLA-Graphene samples. These specimens showed defects in the middle layers of flat-oriented samples  reinforced composites manufactured by FFF technique have been analyzed. The effect of build orientation and graphene reinforcement were studied in particular. Tensile, three-point bending, Charpy impact and interlaminar shear strength tests were carried out to determine the mechanical response of the 3D printed specimens following ASTM standard recommendations. SEM images of ILSS failure samples were evaluated to determine the effects of GNPs on bonding performance.It has been shown that the effect of build orientation was of particular significance on the mechanical performance of PLA-based materials. On-edge and flat orientations showed the highest values for maximum tensile and flexural strengths and stiffness, while upright orientation resulted in the lowest ones. There were no significant differences between PLA and PLA3D850 in terms of tensile and flexural behavior. Furthermore, PLA-Graphene composite samples showed the best performance in terms of tensile and flexural stress and stiffness, except in the case of on-edge orientation. GNPs offered higher stiffness with respect to PLA matrix, preventing the shear strain. More specifically, PLA-Graphene composite showed a significant improvement of the tensile behavior over the other two materials in the case of upright orientation. However, the addition of GNPs tended to reduce the impact strength of the PLA composite samples. Finally, PLA-Graphene composite samples showed the highest interlaminar shear strength. These results were in accordance with the enhanced interlaminar adhesion and performance showed by the PLA-Graphene samples under tensile and flexural loading.Z-axis due to enhanced interlaminar performance. Finally, PLA-Graphene specimens showed, in overall terms, the best performance in terms of surface texture, particularly when parts were printed in flat and on-edge orientations.Moreover, the addition of graphene nanoplatelets did not affect, in general terms, the dimensional accuracy of the PLA-Graphene composite specimens. They showed, in overall terms, the best dimensional accuracy in The results have shown that it is still a challenge to increase the mechanical performance of 3D printed reinforced composite materials with respect to conventional polymer processing methods such as compression or injection molding. A compaction stage after the deposition of the filament would be desirable to reduce porosity. Nevertheless, the properties obtained by 3D printed reinforced composites by FFF are, in general terms, higher than the usual 3D FFF thermoplastics.In conclusion, using FFF to fabricate 3D printed composites with much higher mechanical performance has become a cutting-edge and interdisciplinary research topic in the last few years. It seems to be a very promising technology with potential for future development. It is a relatively new technique and there is a lack of experimental data on the mechanical performance of structures manufactured by this process, underscoring the need for further research to improve our understanding of the mechanical behavior of 3D printed composites.


GaN nanocolumns were synthesized on single-layer graphene via radio-frequency plasma-assisted molecular beam epitaxy, using a thin migration-enhanced epitaxy (MEE) AlN buffer layer as nucleation sites. Due to the weak nucleation on graphene, instead of an AlN thin-film we observe two distinguished AlN formations which affect the subsequent GaN nanocolumn growth: (i) AlN islands and (ii) AlN nanostructures grown along line defects (grain boundaries or wrinkles) of graphene. Structure (i) leads to the formation of vertical GaN nanocolumns regardless of the number of AlN MEE cycles, whereas (ii) can result in random orientation of the nanocolumns depending on the AlN morphology. Additionally, there is a limited amount of direct GaN nucleation on graphene, which induces non-vertical GaN nanocolumn growth. The GaN nanocolumn samples were characterized by means of scanning electron microscopy, transmission electron microscopy, high-resolution X-ray diffraction, room temperature micro-photoluminescence, and micro-Raman measurements. Surprisingly, the graphene with AlN buffer layer formed using less MEE cycles, thus resulting in lower AlN coverage, has a lower level of nitrogen plasma damage. The AlN buffer layer with lowest AlN coverage also provides the best result with respect to high-quality and vertically-aligned GaN nanocolumns. It is also reported that GaN thin-film is achievable on sapphire or amorphous substrates by employing either multi-layer or single-layer graphene as buffer layer14. Such exploitations can be realized by taking advantage of the weak quasi-van der Waals binding17 which can drastically relax the lattice matching condition to be satisfied by constituent materials. That being said, the lack of chemical reactivity of graphene and its extremely low surface energy18 greatly disrupt the nucleation process of GaN, resulting in a low nucleation density19 and difficulty in controlling the stacking sequence of the GaN growth3. The latter issue, which often manifests in the form of stacking faults5 or threading dislocations17, can possibly be suppressed by growing nanowire or nanocolumn structures25. Nevertheless, because of the absence of dangling bonds in graphene, nanocolumns are often aligned in non-vertical directions28 and/or have low density29. Both problems are required to be addressed in order to demonstrate that the growth of III-N semiconductor nanostructures, particularly nanocolumns, utilizing graphene as the substrate can be further considered as an alternative platform in enabling the advancement of optoelectronic device performance and functionality, for instance light-emitting diodes30.Recent progresses in the growth of GaN thin-film on grapheneex-situ manner14. However, the plasma treatments to prepare the growth of GaN on graphene in radio-frequency plasma-assisted molecular beam epitaxy (RF-PAMBE), which is typically done in-situ, often lead to the generation of more severe plasma-induced damage of graphene31. Moreover, it has been evidenced that the GaN nanocolumns may be randomly oriented if the growth is done directly on graphene28. The use of a thin AlN buffer layer can provide substantial protection to the graphene from the direct bombardment of nitrogen plasma and enable the growth of high-density, vertically-aligned GaN nanocolumns on graphene28. Therefore, it is now perceived that the understanding of AlN as an intermediate layer in the GaN/graphene system is necessary to grasp the growth behavior of the GaN nanocolumns in relation to the graphene properties at a given growth condition. Furthermore, it is expected that the insight towards these aspects can shed light on the preference of certain growth conditions over others, which later can be utilized for growing device structures.In this regard, the introduction of defects in graphene by means of plasma treatments could alleviate the issue of absence of dangling bonds in graphene. In case of GaN grown by metal-organic vapor phase epitaxy (MOVPE), the creation of dangling bonds in graphene by this method has been achieved via In the present work, we concentrate on the RF-PAMBE synthesis of self-assembled GaN nanocolumns on transferred single-layer graphene on silica glass for different AlN growth conditions. An insight towards the relation between the AlN migration-enhanced epitaxy (MEE) cycles and the morphology of the AlN buffer layer, as well as the resulting structural and optical properties of the grown GaN nanocolumns on graphene with AlN used as an intermediate layer, is reported. Additionally, the impact of the AlN buffer layer growth on graphene structural properties is presented.first feature is the nucleation of high-density and irregular AlN nanostructures along the line defects of graphene , which is similar to the cases of AlGaN nucleation by MOVPE on graphene29. Commonly, line defects observed in chemical vapor deposition (CVD)-graphene are wrinkles and grain boundaries34. In addition to the surface morphology of the copper foil and the transfer process34, the wrinkles are often related with the compressive stress caused by the difference in thermal expansion coefficients between graphene and the copper foil during cooling from CVD growth temperature to room temperature (RT)35. The grain boundaries are formed as a result of coalesced graphene grains which are grown on the different copper facets during the CVD growth process36. These areas of graphene likely contain defects, which can eventually create dangling bonds to support covalent/chemical bonding of atoms, resulting in a high nucleation density of AlN nanostructures on those particular graphene sites. From bird’s eye-view SEM images, one clearly sees how the nucleation of AlN nanostructures spreads further away from the line defects with the increase of MEE cycles . This will eventually cover the graphene surface, as shown in sample A2, and furthermore in sample A3.The morphology of the thin AlN buffer layer formed on the graphene surface using MEE by RF-PAMBE technique was studied as a function of the amount of AlN deposited, defined by the number of MEE cycles. Details concerning the growth parameters for the AlN buffer layer are outlined in Methods. Three different AlN samples, namely A1, A2 and A3, with respectively 20, 40 and 80 MEE cycles, were grown and characterized. Figures second feature is the formation of individual AlN islands . Unlike the high-density AlN nanostructures along the graphene line defects described above, their size are quite significant, with average diameters of approximately 70, 120 and 200 nm for samples A1, A2 and A3, respectively. In addition to the formation of individual AlN islands, it is also possible for the AlN islands to merge with their nearest vicinity to form coalesced AlN islands . A tendency for AlN to form an island (and its cluster formation) instead of a thin-film could be caused by the extremely low surface energy  of graphene18, which promotes the high surface tension on graphene surface. In addition, the lack of dangling bonds in these graphene surface areas hampers the AlN nucleation process to form a continuous thin-film structure, contrary to the formation of high-density AlN nanostructures in which the dangling bonds are provided by the defects along the graphene line defects. As opposed to this, when the MEE technique was applied to grow AlN on a sapphire substrate at a similar substrate temperature, it resulted in large island sizes and good coverage on the substrate surface37.The first group of features is the row of high-density vertical GaN nanocolumn growth , where nanocolumn diameters vary from 50 to 70 nm. More often, these nanocolumns are found to be grown very close to their neighbor, which is very likely to form coalesced nanocolumn structures, rather than single isolated nanocolumns. Such formation of coalesced GaN nanocolumn structures is clearly visible in sample G1. When formed on 40 MEE cycles AlN buffer (sample G2), it becomes more difficult to discern the rows of vertical GaN nanocolumns, since the density of the nanocolumn structures in general is increased. However, this unique pattern is still observable if one follows the GaN nanocolumns lining with each other, where the nanocolumns have a tendency to coalesce with their vicinity. Interestingly, GaN nanocolumns in sample G3, which has the highest number of AlN MEE cycles, do not meet the expectation of being the most coalesced nanocolumns, forming GaN nanowall-like structures, but rather display nanocolumn structures with identical diameters to those of sample G1.Further RF-PAMBE growth of GaN nanocolumns was then carried out on samples A1, A2 and A3, after which they were re-named as G1, G2 and G3, respectively. In order to gain insight into the influence of the AlN buffer layer on the formation of GaN nanocolumns, the growth conditions of GaN nanocolumns on samples G1, G2 and G3 were kept the same (described in Methods). The representative top- and bird’s eye-view SEM images for samples G1, G2 and G3 are given in Fig. second group of features is the individual vertical GaN nanocolumns . It appears that the number of MEE cycles during AlN formation affects the diameter of the grown nanocolumns, where less cycles give smaller diameter. It is found that the GaN nanocolumns in samples G1, G2 and G3 show average diameters of 90, 130 and 210 nm, respectively. Additionally, it is discovered that there are certain nanocolumns  whose diameter is considerably larger than the mentioned average values. Some of them seem to result from single nanocolumn growth which eventually merges with other nanocolumns in its proximity forming an irregular shape, while there are also circular-shaped nanocolumns where the coalescence process likely starts immediately from the nucleation stage. It should be remarked that GaN nanotubular-like structures (white outlines) can also be formed38 because the edge of the AlN islands can initiate GaN nanocolumn growth39. The third group of features is the formation of tilted nanocolumns . Simultaneous energy dispersive spectroscopy (EDS) and electron energy-loss spectroscopy (EELS) were carried out on the same area to map all present elements. The Ga, Al and O element maps are combined into a color map in Fig. A separate nanocolumn sample was synthesized with nominally the same growth conditions as sample G1 for transmission electron microscopy (TEM) measurements. The cross-sectional high-angle annular dark field scanning TEM (HAADF STEM) image of Fig. A higher magnification of the GaN nanocolumn interface area  of the GaN crystal, it indicates that sample G1 with 20 AlN MEE cycles has the highest density of vertical nanocolumns (since the SEM characterization presented above show that G1 has the lowest average GaN nanocolumn diameter [90 nm] among the G-samples). We note that whereas the peak intensity of the AlN 0002 reflection is higher for sample G2 than for sample G1, there is not much difference in this peak between sample G2 and sample G3, despite higher number of AlN MEE cycles for sample G3 and improved graphene coverage surface area  scans presented in Fig. The ω (rocking) scans of the GaN, shown in Fig. 24. For sample G3, the grown GaN nanocolumns characterized with lower density , (557.9 ± 0.9) and (566 ± 1.0) cm−1. These wave numbers correspond well to the established A1 ), E1 (TO) and E2 (high) phonon modes of wurtzite GaN, respectively53. In line with the former results, the as-grown GaN nanocolumns in samples G1, G2 and G3 likewise exhibit almost identical spectra. As presented in Fig. −1. In addition, the E2 (low) phonon mode  mode at 566.2 cm−1 and its small FWHM  and Si(111)54) correspond to the FWHM of a fully relaxed thick free-standing GaN layer55, which can be an indicator of the high crystalline quality of the GaN nanocolumns. Furthermore, the TO phonon mode at 555 cm−1 related to zinc blende GaN51 is not detected in any of the GaN nanocolumn samples. However, expected A1  and E1 (LO) phonon modes of GaN51 at 734 and 741 cm−1 are not visible in any of the GaN nanocolumn samples, similar to the results obtained by Robins et al.48 and Jeganathan et al.52. Instead, we notice very weak peaks at 715 cm−1 and 743 cm−1  and the 2D band (2680 cm−1) with an intensity ratio of I2D/IG ≈ 2, while the disorder-induced D band (1342 cm−1) is barely seen, making the intensity ratio of ID/IG almost negligible. Changes to the aforementioned intensity ratios, peak positions and possible generation of another band are observed in all of the GaN nanocolumn samples (whereas a faint G* band at ~2456 cm−1 is unaffected). Firstly, the most prominent changes are the decline of the I2D/IG ratio along with an increasing ID/IG ratio from sample G1 to G2 and G3, implying that graphene becomes more defective as the number of AlN MEE cycles increases from 20 to 80. Secondly, another defect-related signature in graphene at 1619 cm−1, known as D’ band56, is noticed only in sample G3. Finally, the peak positions of the D, G and 2D bands experience slight blue shifts, indicating a compressive strain and/or a modification of the carbon atoms in the graphene network by the incorporation of nitrogen atoms which could lead to an n-type doping57. Such blue-shifting peaks have been observed when an intentional plasma nitridation of graphene using either nitrogen58 or ammonia59 causes the introduction of defects as well as changes in the chemical and electronic properties of graphene. Indeed, in this work such nitridation process is an inevitable part of the GaN nanocolumn growth in RF-PAMBE, although during the AlN buffer layer formation the exposure of nitrogen plasma is intended for the nitridation of Al. As shown in Fig. In comparison with the pristine graphene reference sample (i.e. sample before any III-V growth), graphene structural properties after the GaN nanocolumn growth demonstrate quite significant alterations, as can be seen in Fig. e G band 586 cm−1 From the growth observation on the synthesis of GaN nanocolumns on graphene utilizing AlN buffer layers grown via MEE technique with 20, 40 and 80 cycles, it can be suggested that their morphology and orientation are restrained by the AlN structures on graphene. The high surface tension of graphene creates AlN islands on the graphene surface, beneficial for nucleation of GaN nanocolumns. Along with the islands, AlN nanostructures are densely formed along line defects (grain boundaries or wrinkles) of graphene due to the existence of defects which provides dangling bonds. Vertical GaN nanocolumns can be consistently obtained from the AlN islands independent of the number of AlN MEE cycles, whereas the GaN nanocolumns formed on AlN nanostructures can be randomly oriented when they are grown with higher number of AlN MEE cycles. There is also a likelihood of limited direct growth of GaN nanocolumns on graphene, resulting most likely in random growth orientations. Although higher number of MEE cycles produce wider AlN coverage on graphene, the subsequent GaN nanocolumns as well as the graphene property turn out to be not ideal. The structural characterizations and optical measurements present that GaN nanocolumn formation utilizing AlN buffer layer formed with 20 MEE cycles is the most preferable growth condition to obtain overall good quality and vertical alignment of GaN nanocolumns while maintaining the least damage on graphene.2 CVD-graphene grown on copper foil34 and transferred onto the center of a 2-inch supporting silica glass wafer (thickness of 0.5 mm) by Graphene Platform Corp.  was used as growth substrates. All the graphene substrates used in this work refer to an atomic layer of sp2 hybridized carbon atoms arranged in a hexagonal structure with an approximate thickness of 0.335 nm. The samples were grown by an EpiQuest RF-PAMBE system (at Sophia University) equipped with an RF nitrogen plasma source to generate active nitrogen and standard solid-source effusion cells (Knudsen cells) to provide Al, Ga and Si atoms.Commercially available 10 × 10 mm60 at 805 °C, with expectation that the migration length of the supplied atoms on the substrate surface becomes longer than when using the conventional MBE method. Stated substrate temperatures were measured by a pyrometer [ref. –5 Pa, a N2 flow rate of 2.0 sccm and an RF power of 450 W. Each MEE cycle (or MEE period) of AlN consists of four seconds with Al atom supply, five seconds of interruption (resting time) and three seconds with N atom supply27. To investigate the effect of the AlN buffer layer, we grew AlN buffer layer samples A1, A2 and A3 with different layer thicknesses by employing 20, 40 and 80 MEE cycles, respectively. After SEM characterization, these samples were re-loaded into the RF-PAMBE growth chamber and further growth of GaN nanocolumns was performed with conventional MBE method using the following growth parameters28: a Ga flux of 2.5 × 10−4 Pa and a N2 flow rate of 2.75 sccm (RF power of 450 W) at a substrate temperature of 895 °C with a growth duration of 90 minutes. After the growth of GaN nanocolumns on samples A1, A2 and A3, the samples were re-labeled as G1, G2 and G3, respectively.In general, the growth was started by depositing the AlN buffer layer using the MEE techniqueter ref. . MEE wasSEM images were obtained using an SII SMI3050SE focused ion beam-SEM and a Hitachi SU8000 SEM at acceleration voltages of 15 and 10 kV, respectively.The interface of nanocolumn/buffer layer/graphene/silica glass and their element distribution were studied by high-resolution TEM and STEM-EELS in a double Cs-corrected cold field emission gun JEOL ARM200F operating at 200 kV, equipped with a QuantumER GIF for EELS. Details on the TEM specimen preparation can be found in ref. HRXRD was performed with a Bruker D8 Discovery High-Resolution Diffractometer using Cu Kα radiation (1.5406 Å).6 cm−2) was used as the reference sample like for previous studies24.Optical studies were done using RT micro-photoluminescence with a He-Cd laser (325 nm) as the excitation source. A freestanding HVPE-grown GaN bulk substrate (threading dislocation density of 6–8 × 10Unpolarized micro-Raman spectroscopy was conducted at RT in backscattering configuration using a Renishaw InVia Reflex Spectrometer System equipped with a 514.5 nm excitation laser. A pristine graphene transferred onto silica glass was utilized as a comparison to graphene samples after RF-PAMBE growth.


In this paper, we report the finite element method (FEM) simulation of double-clamped graphene nanoelectromechanical (NEM) switches. Pull-in and pull-out characteristics are analyzed for graphene NEM switches with different dimensions and these are consistent with the experimental results. This numerical model is used to study the scaling nature of the graphene NEM switches. We show the possibility of achieving a pull-in voltage as low as 2 V for a 1.5-μm-long and 3-nm-thick nanocrystalline graphene beam NEM switch. In order to study the mechanical reliability of the graphene NEM switches, von Mises stress analysis is carried out. This analysis shows that a thinner graphene beam results in a lower von Mises stress. Moreover, a strong electrostatic force at the beam edges leads to a mechanical deflection at the edges larger than that around the center of the beam, which is consistent with the von Mises stress analysis. The standby power consumption of conventional complementary metal-oxide semiconductor (CMOS) circuits increases to the dynamic ON state level as they are scaled down to the scale of a few tens of nanometers . On the Graphene is a two-dimensional material with excellent mechanical stability and electrical conductivity, and a high Young’s modulus of ~1 TPa . These oIn this work, we present a three dimensional (3D) finite element method (FEM) simulation of double-clamped nanocrystalline graphene beam NEM switches. We focus on designing the graphene NEM switch in line with the experimental work . The pul2 substrate using plasma-enhanced chemical vapor deposition (PECVD) [2- and sp3-hybridized carbon atoms. The deposited NCG film is polycrystalline in nature [L, width W, and thickness t, and a top metal electrode. The schematic representation of the device is shown in In this section, we briefly describe the geometry and operation principles of NEM switches. In our earlier work , we expe (PECVD) . The NCGn nature . The poln nature . The mecn nature . Moreoven nature . The NCGn nature . The poln nature . Usuallyn nature . The pron nature . We usedn nature . For theg0. As the voltage applied between the suspended graphene beam and the top electrode is increased, the resulting electrostatic force balances with the elastic restoring force of the deformed graphene. When the applied voltage reaches a critical point, the electrostatic force overwhelms the restoring force, which causes the graphene beam to be pulled towards the top electrode/fixed element, resulting in closing the switch and thereby leading to a sharp rise in the current flow through the device [The initial air gap of the device is e device . When thThe static electrical and mechanical characteristics of the NEM switch were simulated using the FEM-based CAD tool IntelliSuite  . Figure t = 3, 5, and 9 nm. t = 9 nm, 5 nm, and 3 nm, respectively. This result shows a clear dependence of the pull-in voltage on the scaling of the thickness of the suspended graphene beam. Thus, we confirm that the introduction of thickness scaling leads to a reduction of the pull-in voltage.The von Mises yield criterion is a general way to estimate the yield of any ductile material, such as metals ,24. The NEM switch C has a maximum von Mises stress of 6.2 GPa. When the length of the graphene beam is increased to 1 μm and 1.5 μm, it leads to a decrease in the von Mises stress of 3.8 GPa and 2.3 GPa, respectively. Furthermore, the maximum stress for NEM switch A is 2.5 GPa; when the thickness of the graphene is reduced to 5 nm and 3 nm, the stress is decreased to 1.4 GPa and 0.9 GPa, respectively. V, and the electric field, E, in the free space can be obtained by solving Poisson’s equation [To analyze the impact of the applied electric field on the double-clamped graphene beam NEM switch, we made the same model in COMSOL Multiphysics  . The NEMequation . Figure Z direction at 5 nm above the bottom electrode for the different applied voltages. Consistent with In this paper, we have studied the electro-mechanical switching and mechanical reliability of graphene beam NEM switches by FEM simulations. To evaluate the mechanical reliability of a graphene beam NEM switch, we scaled the length and thickness of the graphene beam and studied the von Mises stress for each structure. This analysis showed that the graphene beam NEM switch with a longer length of 1.5 μm and a thickness of 3 nm has a pull-in voltage of 2 V. The electrostatic force concentration at the edges of the graphene beam leads to more mechanical deflection at the edges than at the center of the beam.


The liquid-phase exfoliation of graphite is one of the most promising methods to increase production and commercial availability of graphene. Because ionic liquids can be easily obtained with chosen molecular structures and tuneable physicochemical properties, they can be use as media to optimize the exfoliation of graphite. The understanding of the interactions involved between graphite and various chemical functions in the solvent ions will be helpful to find liquids capable of dissociating and stabilizing important quantities of large graphene layers. After a step of sonication, as a mechanical precursor, samples of suspended exfoliated graphene in different ionic liquids have been characterized experimentally in terms of flake size, number of layers, total concentration and purity of the exfoliated material. Nine different ionic liquids based on imidazolium, pyrrolidinium and ammonium cations and on bis(trifluoromethylsulfonyl)imide, triflate, dicyanamide, tricyanomethanide, and methyl sulfate anions have been tested. UV-vis, Raman and X-ray photoelectron in addition to high resolution transmission electron and atomic force microscopy have been selected to characterize suspended exfoliated graphene in ionic liquids. The number of layers in the flakes exfoliated, the size and concentration depend of the structure of the ionic liquid selected. In order to obtain large flake sizes, ionic liquids with bis(trifluoromethylsulfonyl)imide anions and a cation with an alkyl chain of medium length should be selected. Smaller cation and anion favors the exfoliation of graphene. The exfoliation caused the formation of C-H bonds and the oxidation of the graphitic surface. Today, two approaches are known to produce graphene: in “bottom-up” methods, such as chemical vapor deposition (CVD), graphene is synthesized, whereas in “top-down” methods, for example liquid-phase exfoliation, graphene sheets are separated from bulk material. The first approach produces low quantities with high quality and large flakes. The second method (top-down) using graphite is low in cost and yields a high concentration of suspended flakes but fabricates limited-size sheets with a low yield of mono-layer graphene. The development of technologies to produce large quantities of high-quality exfoliated graphene is important. One way to achieve a large scalable production is to improve methods of the liquid-phase exfoliation of graphite.Since its isolation in 2004  has stabilized a high concentration of graphene flakes compared to other solvents  are liquid salts below 373 K Seddon, , general4C1im][Ntf2]) and 1-butyl-1-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide . The exfoliation conditions are summed up in 6C1im][PF6]) was also used to exfoliate graphite using an ultrasound technique  has been reported in ILs with promising results. In 2010, Wang et al. (1im][Ntf2]), [C4C1im][Ntf2], 1-benzyl-3-metylimidazolium bromide ([BnzC1im][Br]) and 1,3-bis(phenylmethyl)imidazolium bis(trifluoromethylsulfonyl)imide ([(Bnz)2im][Ntf2]). The concentration of graphene in suspension was measured by UV-vis absorbance spectroscopy. [BnzC1im][Br] has a viscosity that was judged too high to disperse graphite. The concentration of suspended graphene in the [C4C1im][Ntf2] was too low to be detected by absorbance with the author's set up. [(Bnz)2im][Ntf2] stabilized a larger amount of graphene over [BnzC1im][Ntf2] 2im][Ntf2] were found oriented parallel to the surface of graphene unlike the single benzyl substituent in [BnzC1im][Ntf2] im][PF6], was exfoliated using microwaves during 0.5 h. The mixture reached the temperature of 443 K. By proton NMR the authors showed that 10% of the IL was degraded. Graphene had no additional structural defects and 95% of the flakes produced were single-layer graphene. The same protocol was used in [C4C1im][PF6] but with a lower exfoliation rate.In 2015, Matsumoto et al.  was used to analyse the surface showing that the IL intercalated between graphene layers and that the cation preferentially interacts with the surface of graphene compared to the anion. One explanation is that the C2C1im+ cation is not very large and can be interposed between two layers of graphene. Also the interfacial energy between the IL and graphene is comparable to that between two stacked graphene sheets.In 2016, Elbourne et al.  studied in this work are listed in Natural graphite flakes were purchased from Alpha Aesar with a 99.8% purity and a size inferior to 325 mesh. Raman spectroscopy and X-ray diffraction (XRD) were performed, without any treatment, to verify the initial purity  for 24 h. The ultrasonic water bath warmed up during use and reached a maximum temperature of 328 K. This thermal energy expanded the graphite layers and made the exfoliation process easier. As we worked with ILs, this temperature increase did not cause degradation of the solvent. Thus, the water bath was not cooled but was maintained at 323 ± 5 K. The horizontal distribution of the ultrasound is not homogeneous in the bath  solutions were prepared by adding graphite flakes to 2 mL of IL at an initial concentration of 5 mg.mL4C1im][AOT]) from Brown et al. sulfosuccinate  using a MET JEOL 2100FEF (field effect gun energy filtering) microscope with a point resolution of 0.23 nm without removing the IL. First, a thin film of SEG in IL was deposited on a copper mesh 200 grid coated with a layer of formvar/carbon. The grid was then dried gently with a filter paper. The liquid films were observed using an acceleration voltage of 200 kV. For each sample, five squares of the grid were analyzed. High resolution transmission electron microscopy, HRTEM, analysis was also performed on some particles using the same microscope. Subsequently, the HRTEM images and their crystal structure were determined from the diffraction pattern seen in the Fourier Transform images.−1 in the range of 1,200–2,900 cm−1.A volume of 1 mL of SEG was filtered through a polyvinylidene fluoride (PVDF) hydrophobic membrane (from Durapore) with a 220 nm pore size. The membrane was washed with dichloromethane and isopropanol (IPA) to remove the IL from the exfoliated graphite. Then, the filter was dried at 463 K during 12 h. This filter with exfoliated graphite was analyzed by Raman spectroscopy. Raman spectra were recorded at 294 K on a Jobin Yvon T64000 spectrophotometer equipped with a Olympus confocal microscope (100 × objective lens) with a CDD multichannel detector cooled with liquid nitrogen. The excitation source was a 514.5 nm ionized argon laser (Spectra Physics) line. The laser power was adjusted to 200 mW . Spectral resolution is at 0.7 cm2). The Si/SiO2 wafer had been previously washed in IPA during 30 min in an ultrasonic bath. After coating with the exfoliated graphene, the wafer was dried at 463 K for 2 h. The lateral and vertical size of the particules on the wafer were measured by AFM  were casted by spin coating at 1,000 rpm onto a silicium wafer  and Lorentzian (20%) distributions. The analyzed covered a surface of approximately 100 μm2 with a depth of 5 nm.XPS analysis was performed with Versaprobe II PHI 5000(ULVAC-PHI) spectrometer using a 100 μm focused monochromatic Al-Kα X-ray source (1486.6 eV) beam. The high-resolution spectral analysis was performed using a pass energy of 23 eV allowing an energy resolution of 0.5 eV. The XPS spectra were fitted by using Multipak VThe Potentiel of Mean Force (PMF), calculated in this work, corresponds to the reversible work required to peel one layer of graphene from a stack of four layers in different ILs. The method and set up of these PMF calculations by molecular dynamics have been described in a previous paper  among the nine studied ILs. In the ILs with other anions, the concentrations are below 0.5 mg.mL−1 and lower than in ILs with the 4C1im][C(CN)3], [C2C1im][Otf] and [C2C1im][N(CN)2] ILs presented the lowest concentrations of SEG. The relatively high error bars make the distinction between ILs with the 1im][Ntf2] and also in [C4C1im][Ntf2] with a different set up. After exfoliation, no nano-carbon object was found in [C4C1im][Ntf2] and 0.81% of SEG in [BnzC1im][Ntf2]. The low and similar concentrations of SEG reported in these two ILs hinders comparison with our results. Our main conclusion is that using a The [Ci et al.  exfoliat4C1im][C1SO4] which is the IL with the highest viscosity, the concentration of SEG is lower than in the ILs based on the −3. ILs with densities around 1.4 g.cm−3 seem to be favorable to suspend exfoliated graphite, this conclusion being a simple observation and does not necessarily imply causation.In −1 leads to higher concentrations of SEG. In a previous work 3] lead to similar lateral size profiles and to approximately the same amounts of suspended material. We conclude that ILs with the Flakes of exfoliated graphite have larger sizes in [Ntf2], [Ntf2], [C2C1im][N(CN)2], [Ntf2] and [C10C1im][Ntf2] are similar to that of bi-layer graphene, as we can observe a shoulder in According to the literature  distance in the hcp structure. Indeed, it is shown in via AFM depends on the substrate and on the experimental conditions . For example, on Si/SiO2, graphene can show an apparent height of 1 nm 3]. Fifty percent of the exfoliated graphene is in fact graphite with more than 30 flakes while in [C2C1im][Otf], 30% of graphene with <5 layers is found. Using 2C1im][Otf] > [C4C1im][C1SO4] > [BnzmC1im][Ntf2] > [Ntf2] ≃ [C4C1im][Ntf2] > [C4C1im][C(CN)3].The SEG were also analyzed by high resolution TEM to identify monolayers of graphene. The choice of the anion seems important to control the degree of exfoliation of SEG. Adding a benzyl function instead of an alkyl chain on an imidazolium head group will reduce the lateral size of flakes but will favor a higher concentration of graphene.2, sp3 and oxydated carbon atoms at the surface of exfoliated graphite, XPS measurements were performed in samples of washed SEG deposited on a PVDF membrane. Based on references samples studies, we performed C1s core level fitting for exfoliated graphite in different ILs media. In exfoliated graphite, we assigned five main contributions, graphitic carbon (CCgraph) at 284.3 eV, IL related carbon , PVDF related peaks (CCH2 and CF2 at 286.3 and 290.5 eV), carbon sp3 at 284.9 eV and carbon oxyde related peak (C–O and COO at 286.0 and 287.5 eV). 4C1im][Ntf2]. The XPS spectrum of natural graphite is presented in the 3], formation of C-H bonds and oxidation of at least 60% of carbon were found at the surface of the carbon material.The purity of SEG in [Ntf2] was sonicated during 24 h. No differences were found between the XPS spectra of the IL before and after sonication, as shown in the  of peeling graphene in ILs was calculated from MD simulations and is plotted in 4, 1, 1, 1][Ntf2] has demonstrated worst results than [C4C1im][Ntf2], in term of concentration, lateral size and number of exfoliated layers.The PMF curves also indicate that the intercalation of [Ntf2] between two layers of graphene requires more energy than [C4C1im][Ntf2].In In order to attain a more complete view of suspended graphene in ILs, different techniques were used to characterize graphene flakes produced by solvent exfoliation of graphite.The concentration of suspended exfoliated graphene (SEG) is related to the stabilization step. UV-visible spectroscopy measurements reveal that the ILs with 4C1im][Ntf2] and [Ntf2] lead to larger flakes compared to [BnzmC1im][Ntf2] whereas smaller SEG flakes were found in [C2C1im][Otf]. A bulky anion seems to be an advantage to stabilize larger flakes. The number of stacked layers in flakes determined by AFM was smaller in [C2C1im][Otf].AFM and TEM measurements allowed to determine the lateral size of graphene flakes, showing that [C3 hybridization of carbon. [C2C1im][Otf] leads to the highest degree of oxidation compared to the others ILs studied. This result could explain the efficiency of this IL to exfoliate graphite down to single layers. Using an ultrasonic bath to process the exfoliation of graphite does not seems to be the best technique to produce a high concentration of pristine graphene. To optimize the exfoliation process, we need first to increase the reproducibility of the sonication/centrifugation procedure used in this work in order to increase the initial concentration and size of graphite flakes significantly.Exfoliation of graphene in ILs using ultrasound caused surface changes mainly oxydation but also spAdding π-π interactions between the IL and the carbon material does not seem to improve the exfoliation process. With these results, the design of the IL can be optimized by choosing a EB conducted the experiments jointly with BM (TEM), CS (TEM and EIS), DB and P-OB (AMF), AB . MG and AP supervised the project. The manuscript was written by EB, MG, and AP with inputs from all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We studied the effect of low and high sialylated IL4–10 fusion protein (IL4–10 FP) on In vivo activity was assessed in an inflammatory pain mice model upon intrathecal injection.CHO cell lines producing low (IL4–10 FP lowSA) and high sialylated (IL4–10 FP highSA) fusion protein were generated. Bioactivity of the proteins was evaluated in an LPS-stimulated whole blood assay. Pharmacokinetics were studied in rats, analyzing plasma levels of IL4–10 FP upon intravenous injection. in vitro. The pharmacokinetics study showed a 4-fold higher initial systemic clearance of IL4–10 FP lowSA, whereas the calculated half-life of both IL4–10 FP lowSA and IL4–10 FP highSA was 20.7 min. Finally, both IL4–10 FP glycoforms inhibited persistent inflammatory pain in mice to the same extent.IL4–10 FP lowSA and IL4–10 FP highSA had similar potency in vitro and in vivo activity, but clearly results in a difference in systemic exposure. The rapid systemic clearance of low sialylated IL4–10 FP could be a favorable characteristic to minimize systemic exposure after administration in a local compartment.Differential sialylation of IL4–10 fusion protein does not affect the  Interleukin-4 (IL-4) and IL-10, well-known anti-inflammatory cytokines, have potential for the treatment of inflammatory diseases. However, clinical studies with IL-4 or IL-10 have been disappointing ,6. IL4–1in vitro. In whole blood and peripheral blood mononuclear cell (PBMC) cultures IL4–10 FP strongly inhibits the production of LPS-induced pro-inflammatory cytokines such as IL-1β, TNF, IL-6, and IL-8, while having minimal, if any, effect on the production of IL-1RA and sTNFR (ex vivo and in vivo. The fusion protein inhibits pro-inflammatory cytokine production by rheumatoid arthritis (RA) and osteoarthritis (OA) synovial tissue and cartilage cultures  supplemented with 4 mM L-glutamine (Gibco) with pluronic F-68 (Life Technologies), at 0.1 or 0.2%, w/v (culture medium) in a humidified CO2 incubator. Cell number and viability were assessed with a CASY counter (Roche Innovatis AG). Levels of IL4–10 FP produced by the cells were measured in the supernatant with a human IL-10 ELISA kit  according to manufacturer’s instructions.CHOBC® cells were transfected with a proprietary expression plasmid containing cDNA encoding IL4–10 FP, and with a plasmid with cDNA encoding α2,3-sialyltransferase (ST3GAL_IV) using an AMAXA Nucleofector II device (Lonza). After transfection, cells were pooled and seeded at 1.0 × 106 viable cells (vc) per ml culture medium in T-flasks. Cells were incubated for 48 h and seeded in 96-well plates at 300 to 3000 vc/well in culture medium supplemented with Zeocin (Invitrogen) and Blasticidin (Invitrogen) (selection medium). The cells were incubated for ~3 weeks. Visible cell clones were then transferred into 24-well plates containing 2 ml fresh selection medium and screened for IL4–10 FP levels (IL-10 ELISA). Best producing clones were selected, cultured in 6-well plates and expanded into 25 ml T-flasks (4-day culture). Subsequently, they were adapted to culture under shaking conditions and cultured for 7 days while cell density, viability and production were monitored at various intervals (7-day culture). In addition, sialylation of IL4–10 FP produced by different clones was analyzed using lectin-based ELISAs (see below). Finally, two cell lines producing IL4–10 FP with respectively low and high sialylation were selected.To generate IL4–10 FP producing cell lines, CHOBC® clones was evaluated using ELISA-type assays with the galactose-binding Erythrina Cristagalli Lectin (ECL), or the α2,3 sialic acid binding Maackia amurensis Lectin II (MAA). CHOBC® cell supernatant to be tested was diluted in PBS to yield a concentration of 10 μg/ml IL4–10 FP. 100 μl of diluted supernatant was coated in duplicate in the wells of maxisorp plates (Nunc). After O/N incubation at 4°C, plates were washed 3 times with PBS containing 0.05%, w/v, Tween-20 (Merck), followed by incubation with 200 μl/well PBS 0.1%, w/v, Tween-20 (PBS-T), 1 h at room temperature (RT), while gently shaking (300 rpm). 100 μl of biotinylated ECL or MAA (Vector Laboratories), diluted 1 to 10.000 and 1 to 3000 in PBS-T, respectively, were added to the plates, which were subsequently incubated for 1 h at RT. Bound biotinylated lectins were detected by incubation with Streptavidin poly-HRP  in PBS-T for 30 min at RT, followed by incubation with TMB (Invitrogen). The reaction was stopped with 1 M H2SO4 and the absorbance at 450 nm was measured. The ratio MAA/ECL, calculated using the OD450 values of bound MAA and bound ECL, is indicative for the sialylation ratio of the IL4–10 fusion protein.Sialylation of IL4–10 FP produced by the CHOw/v, Bovine Serum Albumin (BSA), and then equilibrated with PBS without BSA. Concentrated conditioned medium containing IL4–10 FP was loaded on the column, which was then washed with PBS. Bound IL4–10 FP was eluted with 0.1 M Glycine, pH 2.25. Fractions were immediately neutralized with 1 M Tris, pH 9.0 and dialyzed in PBS. Fractions containing IL4–10 FP were pooled, assessed for purity and protein content by running a protein gel (see below), a BCA protein assay (Thermo Scientific) and the IL-10 ELISA, aliquoted and stored at −80°C until further analysis.IL4–10 FP was purified from conditioned medium using affinity chromatography according to a previously published protocol . In shorw/v, Tween-20 (PBS-T). The membrane was incubated with the primary Ab mouse anti-human IL-4  or mouse anti-human IL-10 , in 1% milk in PBS-T, followed by HRP-conjugated goat anti-mouse IgG . To visualize the bands ECL Western blotting substrate was added according to manufacturer’s protocol . Stained protein gels and Western blots were imaged using the ChemiDoc MP system (BioRad).Cell supernatants were diluted 1 to 1.33 in Laemmli sample buffer (BioRad), containing 100 mM Dithiotreitol (Sigma-Aldrich), incubated for 10 min at 100°C, whereafter 10 ul was loaded on a 12% polyacrylamide Gel . After electrophoresis, the gel was either stained with Instant Blue (Expedeon) for visualization of total protein content, or prepared for Western blotting in order to specifically detect IL4–10 FP. Blotting was performed using the Trans-Blot system (BioRad) in combination with a 0.2 μm nitrocellulose-membrane Transfer Pack (BioRad), according to manufacturer’s instructions. After transfer, the membranes were blocked in 4% milk  in PBS 0.1%, 2, culture supernatant was collected and tested for TNF concentration with ELISA . The percentage inhibition was calculated relative to TNF production by LPS in absence of IL4–10 FP.Heparinized human blood obtained from healthy volunteers was diluted 1:10 in RPMI1640 medium (Invitrogen), supplemented with 1% penicillin/streptomycin  in 48-well culture plates (Nunc). Lipopolysaccharide  was added at 10 ng/ml, as well as low and high sialylated IL4–10 fusion protein at 0.01–3 nM (0.3–270 ng/ml), final concentrations. As control, HEK293 produced IL4–10 fusion protein was taken along  according to manufacturer’s protocol. Effectiveness of deglycosylation was monitored by Western blotting.1/2 = 0.693*(V/CL), where V is the distribution volume (ml) and CL is the clearance rate (ml/min).Clearance of IL4–10 FP from the circulation was studied in Wistar Crl:WI female rats . The study was approved by the animal ethical committee (project number AVD11500201744) and performed at the animal facility of the Utrecht University. Rats were acclimatized for 1 week prior to the experiment. Rats were intravenously injected via the tail vein with 5 μg IL4–10 FP lowSA or IL4–10 FP highSA, or with a mixture of 5 μg IL-4 (eBioScience) and 5 μg IL-10 (Invitrogen) in a volume of 200 μl PBS. Some rats were injected intravenously with 30 mg asialofetuin (Sigma-Aldrich) in 0.5 ml PBS 10 min prior to injection of low sialylated IL4–10 FP, to saturate the ASGPR. Blood was collected from the tail using a winged infusion system (150 ul per timepoint) in lithium-heparin tubes (Sarstedt) either at 5, 15, 30 and 120 min, or 10, 20, 60 and 120 min after injection of IL4–10 FP and plasma was prepared by centrifugation for 5 min at 2000 g. IL4–10 FP and IL-10 levels were measured with the IL-10 ELISA. IL-4 levels were measured with a human IL-4 ELISA  according to manufacturer’s instructions. Pharmacokinetics were evaluated using non-linear mixed effects modeling (with NONMEM v.7.3). Models were parameterized in terms of fixed effects (population mean) and two levels of random effects; per subject  and per measurement . A one-compartmental model with linear elimination was used as a structural model. Herein, clearance was assumed to be different for IL-10, IL-4, and IL4–10 FP. For IL4–10 FP, the clearance was further differentiated into a slow and fast route, where the fast route represented rapid ASGPR-mediated clearance. The fraction of the dose administered that eliminated via the fast route was estimated separately for high and low sialylated IL4–10 FP. For the rats that received asialofetuin prior to injection with IL4–10 FP, the proportion fast clearance was assumed to be zero. The 95% confidence interval of parameter estimates were calculated by sampling-importance-resampling (SIR). Half-life was calculated using the formula: Tn = 9 for both groups) or vehicle  was injected intrathecally. Mechanical hypersensitivity was measured using von Frey hairs (Stoelting), and the 50% paw withdrawal threshold was calculated using the up-and-down method  as a result of the 4-day culture and 4.2 pcd during the 7-day culture. For CHO372, the specific IL4–10 FP productivity was 3.9 and 3.7 pcd during 4-day culture and 7-day culture, respectively Fig. . These r). Purification of IL4–10 FP lowSA and IL4–10 FP highSA from culture medium using affinity chromatography via an anti-IL4 antibody recovered ~50% of the recombinant protein and yielded ~90–95% purity (data not shown). Both IL4–10 FPs inhibited LPS-induced TNF production in whole blood in a concentration dependent manner, with maximal inhibition at 3 nM . The inhibitory activity was comparable between IL4–10 FP lowSA and IL4–10 FP highSA. Furthermore, both glycoforms inhibited LPS-induced TNF production similar to that of HEK293-produced IL4–10 FP (used in our previous in vitro and in vivo studies)  . These dstudies) . These rIn a pharmacokinetics study we investigated whether the differential sialylation of IL4–10 FP affects its clearance from the circulation. Each IL4–10 FP preparation was injected in rats intravenously (5 μg via the tail vein), and the circulating concentration of IL4–10 FP in serial plasma samples was assessed by ELISA.Concentrations of IL4–10 FP showed an initial rapid decrease upon administration of IL4–10 FP lowSA presumably reflecting fast hepatic clearance Fig. . Adminisin vivo. Intrathecal injection of IL4–10 FP produced in HEK293 cells reduces pain in various mouse models, including models for persistent inflammatory pain . Blockade of the ASGPR with asialofetuin  in rats Although the initial clearance of IL4–10 FP lowSA differed from that of IL4–10 FP highSA, the terminal serum half-life for both IL4–10 FP lowSA and IL4–10 FP highSA were similar. This result is likely due to the presence of a fraction of IL4–10 FP with sialylated galactose-residues in the batch of IL4–10 FP lowSA. Indeed, glycan analysis of IL4–10 FP lowSA, though predominantly showing ECL binding, revealed significant MAA binding too, pointing to the presence of galactose-residues capped with sialic acids.vs 15 kDa). This difference in size impacts the clearance via the kidneys that is mainly affected by the size and the charge of a protein. IL4–10 FP highSA is expressed as a dimer with a molecular weight of ~70 kDa7. Indeed, we observed a considerably increased serum half-life of IL4–10 FP highSA (approximately 21 min), which underlines the contribution of the molecular weight to the clearance rate.Pharmacokinetics in humans of both IL-4 and IL-10 have been studied and indicated a relative short half-life for both cytokines. The apparent serum half-life of recombinant human IL-4 is 19 minutes , while tin vitro potency and different clearance from the circulation. Importantly, both forms effectively reduce inflammatory pain upon local, intrathecal administration. Thus, IL4–10 FP lowSA is an attractive option for local administration as the risk for systemic effects in case of leakage from the local compartment will be minimal due to a rapid clearance by the liver. Conversely, IL4–10 FP highSA might be suitable for systemic therapy since it provides improvements compared to stand-alone IL-10 and IL-4 such as a longer serum half-life and synergistic therapeutic effects. Future studies are warranted to evaluate the clinical potential of either molecule.IL-4 and IL-10 are potent anti-inflammatory cytokines that in preclinical studies yielded promising results ,27. Yet


Introduction: Hepatic granulomas are common in patients with sarcoidosis, but clinically significant liver disease is uncommon and poorly studied. We aimed to characterize the frequency and clinical course of hepatic sarcoidosis in an ethnically diverse population.Methods: This is a retrospective study including all cases of hepatic sarcoidosis in a single center. The median follow-up time was 49 months (4–121). Cases were identified based on ICD-9 and ICD-10 codes for granulomatous hepatitis, sarcoidosis, and hepatic sarcoidosis. The Chi-square and Wilcoxon-signed rank tests were used as indicated to assess for differences between groups.Results: Of 286 patients with sarcoidosis, 27 had hepatic involvement; 78% were female and 48% African American. The most common pattern of liver tests abnormalities was cholestatic. Ten patients had clinically significant hepatic involvement: cirrhosis in seven (25.9%), portal hypertension in nine (33%), and portal vein thrombosis in one (3.7%). Sex, race, and ethnicity were not associated with an increased risk of hepatic involvement or symptomatic hepatic sarcoidosis. Most patients received medical treatment, most commonly oral glucocorticoids. At the end of the follow-up period, all patients were alive but two had undergone liver transplantation due to complications of hepatic sarcoidosis. Three patients with hepatic sarcoidosis had initially been classified as AMA-negative PBC.Conclusions: Hepatic sarcoidosis was found in 9.4% of patients with sarcoidosis and was clinically significant in 37% of those. Identifying and monitoring hepatic sarcoidosis is crucial given its potential complications. Sarcoidosis is a systemic granulomatous disease of unknown etiology characterized by the presence of non-caseating granulomas in affected organs . AlthougMost patients with hepatic sarcoidosis have mild biochemical abnormalities including an elevated alkaline phosphatase (ALP) and gamma glutamyl transpeptidase (GGT) –8. LiverClinical manifestations of hepatic sarcoidosis vary from asymptomatic disease to severe complications due to cirrhosis and/or portal hypertension. The natural history of hepatic sarcoidosis is poorly understood, with important differences reported across diverse racial and ethnic backgrounds. Hepatic involvement is reportedly more frequent and more severe among African Americans. The aim of this study was to describe the frequency of hepatic sarcoidosis and its clinical course in an ethnically diverse population in a single center in the Southeast of the United States and to identify predictors of prognosis among patients with hepatic sarcoidosis.We aimed to identify patients with sarcoidosis and patients with granulomatous hepatitis of all causes. Patients were identified through ICD-9 and ICD-10 codes D86.9 , D86.8 (sarcoidosis of other sites), and K75.3 (granulomatous hepatitis), seen between January 1st, 2010 and December 1st, 2016 at the University of Miami/Jackson Memorial Hospital. After a manual chart review, patients older than 18 years of age with a diagnosis of sarcoidosis or granulomatous hepatitis were included in the study. In addition, those with granulomatous hepatitis were categorized according to etiology, as follows: autoimmune disorders, systemic infections, malignancies, drug-induced, or idiopathic causes.Patients diagnosed with sarcoidosis were further evaluated for hepatic involvement. To confirm the diagnosis of hepatic sarcoidosis, we collected data on the hepatic lesions seen in imaging studies, characteristics of the liver granulomas in biopsies, clinical and/or laboratory evidence of liver involvement, and extra-hepatic manifestations of systemic sarcoidosis. For the purpose of this study, hepatic sarcoidosis was defined as “definitive” if biopsy-proven, and “suspected” when the clinical history and objective laboratory data suggested sarcoidosis and no alternative diagnosis existed.Due to the clinical and histological similarities between primary biliary cholangitis (PBC) and hepatic sarcoidosis, we also retrieved a list of patients with ICD-9 code K74.3 (PBC) in an effort to identify cases of hepatic sarcoidosis potentially misdiagnosed as anti-mitochondrial antibody (AMA)-negative PBC .Data on demographic characteristics, comorbid conditions, extrahepatic manifestations of hepatic sarcoidosis, liver biochemical tests at baseline and at last follow-up, treatment, and clinical outcomes were collected. For patients with AMA-negative PBC, additional information on associated co-morbidities  and bile duct abnormalities on histology were also collected.t-test or Wilcoxon signed rank test. A two-sided p-value < 0.05 was considered statistically significant. Analyses were performed using IBM SPSS Statistics V25.0 .Descriptive statistics were used to summarize the cohort demographics. When appropriate, categorical variables were analyzed using chi-square or Fisher's exact test, and continuous variables using Student n = 326).A total of 632 medical records were identified as having a diagnosis of sarcoidosis and/or granulomatous hepatitis by ICD code. Of those, 286 had confirmed sarcoidosis, 163 AMA-positive PBC, 20 AMA-negative PBC, 3 idiopathic granulomas, 1 tuberculosis, and 159 had insufficient information or incorrect diagnosis, with lack of granulomas, sarcoidosis or PBC . PatientOf the 286 patients with sarcoidosis, 198 (69%) were female, 145 (51%) Caucasian, 122 (43%) African Americans, and 63 (22%) Hispanics. Only 27 had hepatic involvement: 18 with a definitive diagnosis based on biopsy findings and nine with a suspected diagnosis: six were diagnosed based on medical history and three were initially misdiagnosed with AMA-negative PBC which was later modified to hepatic sarcoidosis. Median time from sarcoidosis diagnosis to determination of hepatic involvement was 5.7 months . Median duration of follow-up from diagnosis of hepatic sarcoidosis to last visit was 4.0 years .In our cohort of 27 patients, 20 were female (74%), 13 were African American (48%), 10 were Caucasian (37%) and 4 (15%) were Hispanics. The median age at diagnosis of hepatic sarcoidosis was 45.7 years (range: 23.9–72.5 years). Laboratory tests at baseline and follow-up are illustrated in The most common laboratory abnormality was an elevation in serum ALP, or a cholestatic pattern of liver enzyme abnormalities. Median serum ALP at baseline was 3.5 times the upper limit of normal, associated with minimal elevation in transaminases . Anti-nuAt presentation, the most frequently reported symptom was fatigue, followed by pruritus, weight loss, and hepatomegaly equally . Ten patp = 0.29, 1.0, and 0.62 compared to no treatment 0.29). Antimetabolites elicited a statistically significant change in ALP in individuals with preexisting ALP elevation (p < 0.05). On the other hand, patients receiving oral glucocorticoids or biologic agents showed some lowering in their ALP level which was not statistically significant, p = 0.09, 0.11, respectively.A total of 21 patients were medically treated with one or more agents during our follow-up period; six patients were never treated medically. Oral glucocorticoids were the most frequently used in 17 patients (63%), followed by antimetabolites in 15 patients (56%), and biologic agents in five patients (19%). Median change in ALP from baseline to last follow-up for patients treated with anti-metabolites, steroids, and biologics was −163.5, −219, and −108, respectively, compared to −162.5 in untreated patients  due to complications of hepatic sarcoidosis. The first is a 56-year-old African American woman with well-established cutaneous and pulmonary sarcoidosis, hypertension, hyperlipidemia, depression, and Parkinson's disorder, who initially presented with complications of cirrhosis including esophageal varices, encephalopathy, and hepatopulmonary syndrome. Although the treating physician was concerned about AMA-negative PBC, her explant showed cirrhosis with moderate chronic inflammation, ductopenia, intracellular cholestasis, with multiple portal and lobular non-necrotizing granulomas. There was no evidence of destructive cholangitis. Non-necrotizing granulomas involving the fibroadipose tissue surrounding hilar vessels were noted. Her post-operative course was complicated by delayed graft function, two embolic strokes, and wound infection, but she eventually recovered.The second patient is a 65-year-old Caucasian male with a long-standing 20-year history of extensive granulomatous liver disease which eventually led to end-stage liver disease. He had been treated with ursodiol, multiple courses of steroids and methotrexate without success; he underwent OLT due to complications of portal hypertension. His explant showed a cirrhotic liver with marked cholestasis, ductopenia, focal periductal fibrosis, mild chronic inflammatory infiltrates, and non-caseating granulomatous inflammation. The post-operative course was remarkable for partial wound dehiscence, an episode of cholangitis, and acute cellular rejection.p = 0.66, 0.79, 0.15, respectively). In our cohort there was no statistically significant association between sex (p = 0.49) or race (p = 0.86) and clinically significant hepatic sarcoidosis.Sex, race, and ethnicity were not associated with an increased risk of hepatic involvement in patients with systemic sarcoidosis ; in addition, both can coexist with sarcoidosis , 21. In In conclusion, our study highlights the importance of correctly identifying hepatic sarcoidosis, given increased morbidity when associated with cirrhosis and/or portal hypertension. Race, sex, and ethnicity may not predict hepatic involvement and disease progression. When appropriate, special attention should be given to AMA-negative PBC patients to exclude the possibility of hepatic sarcoidosis.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by University of Miami Institutional Review Board. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.CL designed the study concept. Data collection was performed by NF, PS, and LD. Analysis and interpretation of data was performed by MS and CL. Draft of the manuscript was performed by NF and MS. MG-B, MM, and CL provided the critical revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.