The choice of the right material is essential in microwave processing. The carbon materials are good microwave absorbers, which allows them to be transformed by microwave heating into new carbon materials with adapted properties, capable of heating other materials indirectly. In this paper, the microwave heating of graphene as reinforcement of the lithium aluminosilicate (LAS) ceramics has been explored. LAS ceramics have a near-zero coefficient of thermal expansion and exhibit an effective and efficient heating by microwave. Nevertheless, we have found that the graphene did not show any significant response to the microwave radiation and, hence, the interaction as mechanical reinforcement with the LAS material is harmful. The possible benefits of graphene materials to microwave technology are widely known; however, the mechanism involved in the interaction of microwave radiation with ceramic-graphene composites with high dielectric loss factors has not been addressed earlier. The graphene oxide (GO) was supplied as slurry (3.2 wt % in distilled water).β-eucryptite powder were added into 100 mL of water and it was dispersed under continuous stirring for 30 min. Water suspensions of GO with two different contents (0.5 and 1 wt %) were gradually added into the 10 g of β-eucryptite suspensions under continuous magnetic stirring and then sonicated for 30 min in an ultrasonic probe . The final suspension was frozen in a liquid nitrogen bath and then dried in a freeze drier  for 24 h. The dried powder mixture was ground and sieved using 60 mesh.10 g of 111 mode with a resonant frequency of 2.45 GHz was selected as the heating cell for microwave. The electric field vectors are perpendicular to the cavity axis with the maximum electric field magnitude at the center, where the samples are located. LAS-GO powders with 0.5 and 1.0 wt % of GO were sintered under vacuum using this microwave technology . Cyl. Cyl111 2, H2O and hydrogen sulphur as SO2. The oxygen content was determined using a graphite furnace coupled with LECO VTF 900 equip. In total, 1 mg of LAS-GO powders composite was pyrolyzed to 1350 °C under a helium flow of 225 mL·min−1. The generated CO oxidizes to CO2 forming CuO, which was evaluated and thereby the oxygen content was obtained directly.The carbon, sulphur, hydrogen and nitrogen content were determined by combustion of 1 mg LAS-GO powders at 1050 °C . Under these conditions carbon is transformed into COThe composites sintered at different temperatures were found to have the following graphene contents: 0.45 and 0.90 wt % to LAS + 0.5 wt % GO and LAS + 1.0 wt % GO, respectively.Raman spectra were recorded using a Confocal Raman Microscope  with a 532 nm excitation laser. Up to 20 spectra were recorded along the whole thickness of the polished composites. The sintered samples were previously cut longitudinally in half cylinders with a diamond saw and polished  to 0.25 μm using SiC paper and diamond suspension.−3 for LAS and graphene, respectively. The relative density was calculated by dividing the bulk density with the theoretical density of the powder mixture.The bulk density of the samples was measured by the Archimedes method (ASTM C373-88) with ethanol as the immersion medium using densities of 2.39 and 2.01 g·cmNanomechanical properties such as hardness and Young’s modulus of the sintered composites were obtained by the nanoindentation technique . To carry out indentations at very low depths, a Berkovich diamond tip was used with radius less than 20 nm. In order to ensure the quality of the tip throughout the experiment, pre- and post- calibration procedures were performed for this indenter ensuring the correct calibration of its function area and correct machine compliance. The nanomechanical properties of the sintered samples were evaluated from the load-displacement nanoindentation data using the widely accepted Oliver and Pharr model .The fracture surface sections and microstructure of the sintered samples have been studied using a Field Emission Gun Scanning Electron Microscope . Specimen microstructures were thermal etching during 30 min at 100 °C below the maximum temperature to reveal grain boundaries. The average grain sizes were measured using the linear interception from FE-SEM micrographs. Approximately 100 grains for each phase were studied [A complete Raman spectroscopy study was performed to evaluate and optimise the graphene thermal reduction by microwave radiation. Raman spectroscopy is a very useful technique in order to evaluate the thermal reduction of the graphene oxide (labelled as rG) along the composite.Electrical resistivity values of LAS and LAS-rG materials are summarized in Density values were very high and close to the theoretical values for monolithic LAS materials . HoweverH) and Young’s modulus (E) values as a function of penetration depth.Some mechanical tests were performed in order to study the behaviour of the composites obtained by microwave processing. The nanoindentation technique shows a common trend clearly detected in the behaviour of hardness  of the samples sintered by microwave at 1200 °C and 1250 °C is presented in 2g phonon of sp2 C atoms (G peak) and the breathing mode of k-point photons of A1g symmetry (D peak), respectively, were observed in all the samples [−1 and an additional peak at 2945 cm−1 ascribed to the G + D combination mode induced by disorder or the D + D’ band [ samples . The eff D’ band . The resAs a result of the reduction process, non-conductive graphene oxide was transformed into a conductive material. Electrical resistivity values of LAS and LAS-rG materials are summarized in The addition of very small amounts of graphene into the LAS matrix leads to an exponential increase in the electrical conductivity of the composite in comparison to the monolithic LAS.The percolation threshold of the as prepared composites was found to be around 1 wt %, therefore, when the graphene content increases there is an increase in the intersheet connections. In the case of CNTs or CNFs, quantities around 7–10 vol% are needed in order to make the ceramic composites electrically conductive . The conThe value of electrical resistivity obtained (~4 Ω·cm) with LAS + 1 wt % rG sintered by microwave (in both temperatures), is good enough to shape the nanocomposite using the electro discharge machining (EDM) technique ,34. ThisCarbon materials are, in general, very good absorbents of microwaves, i.e., they are easily heated by microwave radiation. This characteristic allows them to be transformed by microwave heating to give rise to new carbon materials or to be used as microwave receptors, in order to heat other materials indirectly, or to act as catalysts and microwave receptors in different heterogeneous reactions. As a consequence, in recent years, the number of processes that combine the use of carbon materials and microwave heating has increased in comparison to other methods that are based on conventional heating ,18.The blank hypothesis to be tested in this work is that graphene could have a great influence on the microwave sintering behavior of LAS composites, and therefore, this may mean an increase in their final density, even at a lower temperature. In previous works, it was found that the LAS is an excellent material to absorb the microwave energy and, on the other hand it is known that the graphene has a high dielectric loss factor compared with the LAS ceramic ,36.From the results obtained, it is clear that graphene as a second phase directly affects the resulting densities of the studied LAS composites. This can be due to some important factors: the poor adherence of graphene with the LAS matrix, the lack of heat transfer by conduction from the graphene to the ceramic matrix, the agglomeration of graphene or the absence of the microplasma effect . We willThe microplasma effect would cause an increase in the local temperature of the graphene platelets. Thus, graphene platelets should be heated rapidly due to (a) high absorption of microwave radiation and (b) transfer of heat to the LAS matrix. Due to the nature of volumetric heating of microwaves, all the graphene platelets might simultaneously absorb microwaves and the temperature of the LAS particles that surround the graphene should also increase. Hence, the heating rate of the mixture would be far better than the one of the pure components, and the positive result (and expected) will be an increase of the density values even at lower temperatures than the one of the dense LAS material (1200 °C). This evidently has not happened. Therefore, the graphene is not acting as a susceptor during the sintering process, as initially thought.To verify this fact, the sintering of the same material using conventional heating at 1200 °C in vacuum with 2 h of holding time has been carried out. The obtained density values were: LAS (89.5%), LAS + 0.5 wt % rG (90.2%) and LAS + 1.0 wt % rG (91.3%). The density values were very low for all the materials, even the LAS composites with graphene that showed similar values to the ones obtained by microwave processing. Therefore, it has been shown that despite having a high dielectric loss factor and be an excellent electrical conductor, graphene does not act as a good susceptor and does not improve the sinterability of the matrix. An important conclusion is that there was no effect due to the microplasma generated by the graphene in a microwave environment.H) and Young’s modulus (E) with penetration depth. The large dispersion of H and E for the initial 200 nm of penetration depth could be due to the implicit experimental variability of factors such as tip-sample interactions, sample roughness and tip rounding. As expected, the low densities recorded for the different composites, are reflected in a sharp drop in the mechanical properties compared to those achieved in monolithic LAS materials. At 1200 °C, the composites with 0.5 wt % rG, the mechanical values tend towards the same value with the depth test. However, composites with 1 wt % rG, both E and H show a greater dependence with the penetration depth and sintering temperature. Increasing the sintering temperature up to 1250 °C, results in a slight improvement of the mechanical properties, closer to the monolithic LAS. In both cases and keeping in mind the density values, is possible to affirm that the graphene as a second phase in composites leads to a decrease in the mechanical properties, proportional with the quantity of graphene. These data also confirm that graphene is not acting as a susceptor during microwave heating.The next figure  draws thβ-eucryptite grain size reached depending on the presence of graphene, and their percentage within the matrix. The grain size of LAS decreases with the graphene content, i.e., graphene inhibits the grain growth of the matrix, and this grain in the composites is less defined in comparison to the monolithic material. The grain size of the starting powder of LAS is, approximately, 1.0–1.5 µm and the β-eucryptite grain size reached in the monolithic material by microwave is less than 2 µm [han 2 µm ,8Up to now, the graphene that should be the responsible for the microwave absorption due to a high dielectric loss factor and so, generate heat and heat the other constituent(s) by conventional modes of heating, is the one that causes the poor adherence between the graphene sheets and the ceramic matrix leading to poor mechanical properties in the composites. A possible explanation, which should be thoroughly studied, in order to explain the bad results obtained in the properties of the composites sintered by microwave radiation, is the change in the crystal structure of graphene when a perpendicular electric field was applied . Some auThis is a supposition or proposed explanation made on the basis of limited evidence as a starting point for further investigation.The 7 K−1 is obtained and 160 °C to 300 °C the value drops to −1.208 × 109 K−1. Therefore, it would be expected that adding graphene in a matrix could lead to more negative CTE values.Su et al. , measureCTE values for composites obtained by microwave technology do not exhibit the expected behavior. In the case of the composites with 0.5 wt % of rG, the CTE values are slightly lower than those obtained for the LAS monolithic material for all the sintering temperatures. In the case of composites with 1 wt % of rG, the CTE values present the same order as those obtained for the LAS material.The study of the behavior of graphene as a second phase in LAS matrix composites obtained by a non-conventional microwave sintering technique has been carried out for the first time, to the best of our knowledge, in the current investigation. The graphene oxide has been reduced in a one-step by microwave radiation, and therefore, the LAS-rG composites show a high electrical conductivity with very low graphene contents. The dilatometric data, including cryogenic temperatures, show a controlled CTE with very low thermal expansion behavior at all temperatures. The addition of graphene as a second phase, leads to near-zero values. These characteristics could lead to a variety of ceramic materials to suit engineering applications.Nevertheless, the possible potential of graphene to act as a susceptor and help to heat others dielectric materials by microwave technique is null. The mechanisms and effects involved in the graphene during the application of a perpendicular electric field are still unknown. From these results it can be concluded that graphene, despite having a high dielectric loss factor and being a good candidate to contribute in the sintering process, does not improve the density and the final mechanical properties such as hardness and Young’s modulus of LAS-rG composites. This work will enrich the current graphene-microwave knowledge.


The effects of the wet milling rotating speed on the number of graphene layers and graphene quality, and the conversion efficiency of graphite exfoliate to graphene, were investigated by scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The results show that the number of few-layer graphene nanometer sheets (GNSs) (≤10 layers) gradually increases with the increase of rotational speed in the range of 160–240 rpm. The proportion of GNSs with 0–10 layers reaches more than 80% as the rotational speed is increased to 280 rpm. GNS defect types in the composite materials are marginal defects with minimal influence and almost no oxidation. In the range of 160–280 rpm, the intensity of graphite peak decreases and the conversion efficiency of graphene increases with the increase of rotational speed. This is the same as the experimental result obtained by HRTEM. Currently, graphene has great potential applications and is widely used in aerospace, electronics, energy, structural and mechanical engineering, environment, medicine, and packaging applications. The nano-graphene core-shell structure is an important research direction for the comprehensive performance and multiple functions of composite materials .4C as a sintering aid, added 0–5.0 wt.% GNSs as experimental raw materials, and sintered at 2130 °C for 1 h without pressure to prepare GNSs/SiC composites [x) under CmSn  conditions [8C12 in the gas phase measured by the infrared resonance enhanced multiphoton ionization technique with X = V, Nb, Zr based on first principles [2 to prepare graphene and one-dimensional graphite nanoscrolls. This study shows that graphene can be effectively obtained by mechanically shearing and exfoliating graphite in a certain medium. Ouyang et al. used plasma-assisted dry ball milling to promote the exfoliation of graphite and reduce its disorder [At present, the solid lubricant fillers used frequently mainly include graphite, carbon nanotubes, and graphene, but they have the disadvantages of low hardness and toughness. By combining solid lubricant with hard ceramic materials, the defects of low toughness and low thermal conductivity of ceramic particles can be remedied so that the lubricating effect of soft particles and the strengthening effect of hard particles can play a role together . The wramposites . Tang etmposites . When thmposites . Howevernditions . Gueorguinciples ,15 and Ginciples  developedisorder . Gorrasidisorder . Zhang edisorder . Comparedisorder . At presIn this paper, graphene-wrapped SiC nanocomposites were synthesized by wet milling method using graphite and SiC as raw materials . The effThis article is divided into three parts: introduction, materials and methods, and results. The introduction mainly collects the related research on graphene-wrapped composite materials in recent years and puts forward the research purpose of this article. The material and method part mainly explains the initial materials and specific steps of the experiment, and introduces the role of the relevant instruments in the experiment and specific parameters. The results part mainly summarizes and discusses the experimental results of SEM, HRTEM, Raman spectroscopy, FTIR, and XRD.3N4) milling ball was used as milling medium with a diameter of 5 mm, and the process control agent is ethanol and distilled water.The size of graphite flakes is about 10 μm, and for SiC nanoparticles, is about 50 nm. A silicon nitride  and distilled water solution (volume ratio = 7:3) are added to the milling jar as an abrasive medium or process control agent.The milling jar is clamped in the planetary ball mill with the clamping fixture, and the machine cover is covered. The rotating speed is set at 160 rpm, and the milling time is 50 h.When the wet ball milling is finished, the powder is dried in a vacuum drying oven and then tested.The rotational speed of the ball mill is adjusted to 200, 240, and 280 rpm, and the above steps repeated for the experiment.Graphene-wrapped SiC core-shell nanoparticles were synthesized using a planetary ball mill  at 220 V voltage and 16 A current in one step. The movement mode of the planetary ball mill is different from that of the ordinary ball mill. As shown in −1. The chemical bonds and functional groups of the composites were detected using a Fourier transform infrared spectrometer  in the spectrum range of 750–4000 cm−1, which provided a favorable basis for the quality detection of graphene.The powder after wet ball milling is dried in a vacuum drying oven . The initial materials before ball milling and the surface morphology of the composites after ball milling were observed by high resolution scanning electron microscope  at 5 kV voltage. The dispersion of graphene-wrapped SiC core-shell nanoparticles and the number of graphene layers at different rotational speeds were observed by high resolution transmission electron microscope  at 200 kV. For the detection of the conversion efficiency of graphite exfoliation to graphene, the X’Pert Powder X-ray diffractometer  was used to conduct qualitative and quantitative analysis of the composite material within the range of 2θ from 10–80°. The defect types and sizes of carbon-related materials were evaluated by Raman spectrometer  in the spectral range of 1000–2000 cmThe HRTEM morphologies of graphene-wrapped SiC nanoparticles at 160 and 280 rpm were observed  as shown in 6 times magnification) morphology of graphene-wrapped SiC nanoparticles at different speeds were randomly selected. From this figure, the graphene-wrapped SiC core-shell nanoparticle microstructure can be seen clearly. Single-layer graphene thickness is 0.335 nm and the number of layers of multilayer graphene can be calculated by HRTEM. It can be seen from As shown in Rafael Tadmor deduced the mathematical expression of the interaction energy between macroscopic objects with common geometric shapes by establishing the London–van der Waals interaction energy among different geometries , which pA is the Hamaker constant (2.38 × 10−19 j).This model can be compared to the graphite flakes and single-layer graphene in this experiment. When the shear force between the graphite flakes given by the milling ball is greater than the van der Waals force between the graphite flakes, the graphene will exfoliate from the graphite flakes. The calculation formula is as follows :(1)EA=−Ah1, h2 is the thickness of single-layer graphene at 0.335 nm, and d is the distance between two graphite layers at 0.335 nm. The calculation shows that the energy required to exfoliate a single layer of graphene from the graphite flake is 4.2 × 10−13 j. According to this method, the energy required for exfoliate 10 layers (3.35 nm) and 20 layers (6.7 nm) can be calculated.Most of the graphite flakes used in this experiment are 10 μm, which was set as the thickness of mmρ—The density of Si3N4 milling ball is 3100 kg/m3;The energy transfer from the milling ball to graphene was evaluated with the increase of ball milling speed. The calculation formula is as follows :(2)△E=π1mmd—The diameter of the milling ball is 5 mm;vmm1—The tangential velocity vmm1 of the faster milling media is 0.85 m/s;mm2v—The tangential velocity vmm1 of the slower milling media is 0.425 m/s;PY—The Young’s modulus of the final product is 230 GPa; mmY—The Young’s modulus of the milling media is 388 GPa.−8 j, which is enough to exfoliate all sizes of graphite into graphene. While ensuring that the structure of the graphite flake is not destroyed, as the speed of the ball milling increases, the tangential speed of the milling media is also constantly increasing. Therefore, the energy transferred from the milling media to the graphene is also increasing. When the ball milling speed is faster, the graphene will be exfoliated into fewer layers.The energy provided by the milling medium is 9.67 × 10The thickness of the produced GNSs was estimated from the number of layers of the GNSs in their TEM images. The layer number distribution of GNSs was obtained from 40 random HRTEM images, either standing alone or wrapping nanoparticle. As shown in In the process of counting the number of graphene layers, it was found that the number of GNSs that can be wrapped on SiC nanoparticles is often 7 layers or less, and the thick GNSs are almost all scattered in the composite material. Shown in The performance of graphene in tribological behavior is not only affected by the number of layers, but also related to the types of defects in grapheme. Wang et al. used molecular dynamics method to study the effect of vacancy defect and SW defect on the friction force of graphene. Simulation results show that the friction on graphene with both kinds of defects were obviously larger than perfect graphene in the defect regions, which was attributed to the increasing energy dissipation resulted from the extended interface barrier potential of graphene due to vacancy . Therefo−1), G peak (1580 cm−1), and D’ peak (1620 cm−1) [Shown in 20 cm−1) . 2 carbon atom and its interaction with iTO and iLO optical phonons promote the generation of G peak, which is a very rare Raman scattering process in graphene. As the D peak and G’ peak of the second-order double resonance Raman scattering process, iTO optical phonons will be generated under two inelastic scattering between valleys. Among them, the D peak is related to the scattering between iTO phonons and defect valleys, and the number is one. According to experience, the appearance of the D peak is caused by the deviation of the lattice of the graphite flake layer from the Brillouin zone, so the D peak reflects the defects of the graphene lattice. The G peak is due to the in-plane vibration produced by the σ bond bonding of carbon atoms [The vibration direction of the atom parallel to the plane of graphene is expressed as (i), the vibration direction perpendicular to the plane of graphene is expressed as (O), the vibration direction parallel to the direction of A–B carbon bond is expressed as longitudinal (L), and the vibration direction perpendicular to the direction of A–B carbon bond is expressed as (T). The in-plane vibration of spon atoms . D/IG is a recognized index for evaluating the strength of defects in carbon-related materials [D/IG intensity ratio of initial graphite is about 0.03, and the ID/IG intensity ratios in the experiments at 160, 200, 240, and 280 rpm are 0.41, 0.57, 0.59, and 0.68, respectively. Its value is much lower than that of the graphene prepared by redox method (ID/IG > 1) [D/IG > 1.5) [D/IG strength is related to the gradual thinning and exfoliation of graphite flakes into graphene and the generation of defects. There is no obvious D’ peak in the experiment with the rotational speed of 160 rpm. When the rotation speed is increased to 200 rpm, a faint D’ peak appears, which is a small part of the G peak and gradually increases with the increase of the rotational speed. According to the ratio of ID/ID’, information about the nature of the defect can be obtained. The defect types can be divided into sp3 hybridization-related defects (≤13), vacancy defects (≤7), and edge defects (≤3.5) [The intensity ratio Iaterials ,37. As c/IG > 1)  and dry G > 1.5) . From ths (≤3.5) . Accordi−1, and there is no C=O, –OH stretching vibration peak, and C–O–C vibration absorption peak related to graphene oxide [−1 are the absorption peaks of CO2 in the air.ne oxide ,41. Two It can be seen that the graphene conversion efficiency of the composites is very high after 50 h milling. With the increase of milling speed, the conversion efficiency of graphene increases gradually. At the same time, it can ensure the integrity of SiC nanoparticles to a certain extent.The conclusions obtained using SEM, HRTEM, Raman spectroscopy, FTIR, XRD, and other characterization methods for graphene-wrapped SiC core-shell nanomaterials may be equally applicable to other similar graphene-wrapped inherently nanostructure systems.In this paper, the rational speed of the ball milling was changed by wet ball milling, and the influence of different rational speeds on the number of graphene layers, quality, and conversion efficiency was analyzed.The results show that the graphene is well dispersed in SiC nanoparticles when the ball milling speed is in the range of 160–280 rpm, and graphene-wrapped SiC core-shell nanocomposites were synthesized.With the increase in ball milling speed, the proportion of few-layer graphene (≤10 layers) increases. When the rotational speed is 160–240 rpm, the proportion of few-layer graphene increases from 45% to about 70%. When the rotational speed is increased to 280 rpm, the proportion of few-layer graphene increases to more than 80%. At the same time, it is found that most of the GNSs wrapped on SiC nanoparticles are ≤7 layers.When the rotational speed is 160–280 rpm, the obtained graphene has relatively small defects, and the defect type is the edge defect that has the least impact on the quality of the graphene and, at the same time, it is almost not oxidized. Therefore, the graphene produced by wet ball milling has better quality.With the rotational speed increases, the peak intensity of graphite gradually decreases and the conversion efficiency of graphene gradually increases. When the rotational speed reaches 280 rpm, the conversion efficiency of graphene has reached an ideal state.The graphene-wrapped SiC core-shell nanocomposites prepared by wet ball milling have high quality. When there are more graphene layers, the anti-friction ability and self-lubricity are stronger. Therefore, in practical application, when the main purpose of the required materials is to reduce friction and wear, a lower ball milling rotational speed can be selected. If the composite material is for the purpose of improving the toughness, thermal conductivity, and optical properties, the ball milling rotational speed can be appropriately increased.


Meanwhile, the electro‐enhancing influence of OMe/OH groups could be switched off or inverted by conformational twisting. 267 conformational equilibrium constants measured across eleven solvents were found to be better predictors of reaction kinetics than calculated electrostatic potentials, suggesting utility in other contexts and for benchmarking theoretical solvation models.The description of substituents as electron donating or withdrawing leads to a perceived dominance of through‐bond influences. The situation is compounded by the challenge of separating through‐bond and through‐space contributions. Here, we probe the experimental significance of through‐space substituent effects in molecular interactions and reaction kinetics. Conformational equilibrium constants were transposed onto the Hammett substituent constant scale revealing dominant through‐space substituent effects that cannot be described in classic terms. For example, NO The description of substituents as electron donating or withdrawing leads to a perceived dominance of through‐bond influences. The situation is compounded by the challenge of separating through‐bond and through‐space contributions. Measurements of through‐space substituent effects question classic descriptions of substituent effects. Similarly, the computational prediction of solvent effects remains notoriously difficult.19F NMR signals corresponding to each conformer. The integral ratio corresponds to the conformational equilibrium constant, KX, which constitutes a quantitative assessment of the substituent effects on interactions occurring within the balances. Negative electrostatic potential over the X‐substituted ring would repel the electron‐rich carbonyl oxygen and contribute towards a preference for the H‐conformer in Figure KX for the 25 molecular balances shown in Figure 6]benzene , encodes the electronic effects of the X substituent on the position of the conformational equilibrium. These −log10(KX/KH) values are accordingly positive for classically “electron‐withdrawing” para‐substituents  and negative for “electron‐donating” para‐substituents . Such substituent effects are reflected in the calculated electrostatic potentials taken over the carbon positioned ipso to the conformationally exchanging formamide  for all 25 molecular balances in benzene ). The 3,5‐dinitrophenyl substituent in compound 1‐e =+1.00. Such behavior would classically be described as more electron‐withdrawing than the directly connected nitro substituent in compound 21‐NO (σp(conf)=+0.90). Strikingly, moving the two nitro groups such that they are positioned over the biaryl bond in compound 1‐d, results in a very large change in the determined Hammett substituent constant (σp(conf)=−0.17, orange in Figure σp(conf) can be attributed to the addition of a tert‐butyl group, but the σp=−0.20 for a tert‐butyl group,σp(conf) of −1.17 between 1‐d and 1‐e.The experimentally determined conformational equilibrium constants can be transposed onto the established σs Figure .2, 21 Heipso=−66 vs. −16 kJ mol−1, Figure 1‐d. Indeed, despite the large electrostatic change arising from differences in the proximity of the nitro oxygen atoms, these ESPipso values remain excellent predictors of the conformational preferences of the balances  provide insight into the large changes in substituent effects upon repositioning the nitro groups Figures  A,B. Theσp(conf)=−0.85, it might be tempting to describe the 2,6‐dimethoxyphenyl substituent in 1‐a as being even more “electron donating” than the amino group in 21‐NEt (σp(conf)=−0.66, red in Figure 1‐b (σp(conf)=+0.03, green in Figure ortho tert‐butyl groups, which sterically twist the OMe groups such that the oxygen lone pairs no longer point over the adjacent phenyl ring. Accordingly, the ESPipso value over the bridging phenyl ring in 1‐b was similar to the control compound 1‐H, which does not contain any OMe substituents  from +0.03 to +0.54 =+0.47). The electrostatic slices in Figure 1‐c.Remarkably, removing the capping methyl groups from compound 1‐a to 1‐c, ESPipso on the bridging ring changes by 78 kJ mol−1 and σp(conf) changes by ≈1.4 units =−0.11 and −0.37 vs. −0.17 and −0.85). Again, the negative sign of σp(conf) for the ortho‐nitrophenyl substituent runs counter to the traditional expectation that a nitro group is strongly electron withdrawing.Hammett constants determined in the mono‐1‐n) gave σp(conf)=+0.11, with the through‐space δ+ charge of the NH protons overcoming through‐bond resonant donor ability of the nitrogen lone pair .Positioning an amino group over the biaryl bond (meta‐methoxyphenyl substituent in 1‐k (σp(conf)=−0.31) exhibited an electro‐enhancing influence even though meta‐methoxy groups normally have net electron‐withdrawing character (σp=+0.12).meta‐nitrophenyl substituent (1‐j). However, ESP slices reveal that the bridging phenyl ring is located in the electro‐attenuated region when bonded to a meta‐nitrophenyl group, but in the electro‐enhanced region for the meta‐methoxyphenyl example .The  σp=+0.1.2 Surpripara‐substituted phenyl rings of balances 1‐h and 1‐i project their NO2 and OMe substituents along the same axis as the simple control balances 21‐NO and 1‐OMe. Accordingly, the signs and magnitude of their respective σp(conf) values (+0.72 and −0.15) are consistent with their substituent effects being projected in the same direction, but over a greater distance than the aforementioned control balances (σp(conf)=+0.72 vs. +0.90, and −0.15 vs. −0.25).The ortho to the biphenyl bond (1‐l and 1‐m) gave similar substituent effects to the control compounds 1‐H, 1‐Me, and 1‐Ph. Such findings also rule out electronic changes arising from “steric pressure”.Apolar methyl and ethyl groups positioned ipso values utilized in Figures 2=0.94, Figure S1D). Similarly, the same through‐space electrostatic potentials, in which through‐bond contributions are definitively absent, also correlated well with the experimental conformational equilibrium constants .Our observations are in accord with Wheeler and Houk's proposal that through‐space field effects, and not through‐bond polarization of the aryl π‐system, are the major determinants of non‐covalent interactions involving aromatic rings.Having convinced ourselves of the dominance of through‐space substituent effects over through‐bond polarization of the aryl π‐system, we next sought to examine whether such dominance is manifested in other contexts; namely solvent and reactivity influences.Ka values of benzoic acids in aqueous solution), even though the electronic effects of substituents can be modulated by the solvent.10(KX/KH) values for 267 substituent/solvent combinations were determined using 19F NMR spectroscopy spanning 25 molecular balances in eleven different solvents (Table 10(KX/KH) values of the simple control balances appear to be relatively insensitive to the solvent  values for the most polar substituent/solvent combinations deviate most strongly from the 1:1 line. For example, outliers in dimethylsulfoxide (DMSO) include compound 1‐a (red point), and those containing good H‐bond donors  differences in the intramolecular interactions between the O‐ and H‐conformers (ΔE), and ii) the change in Boltzmann averaged hydrogen‐bond donor and acceptor constants between each conformer (Δα and Δβ), as represented in Figure G=−RTlnKX as the αs and βs solvent H‐bond constants were varied) were fitted to the equation in Figure 2=0.85). Pleasingly, the dissected solvent‐independent ΔE values (Table S10) gave an improved correlation against the electrostatic potential over the ipso‐carbon  compared to the prior correlation against equilibrium constants determined in [D6]benzene . The improved correlation reaffirms the dominance of electrostatics in determining the conformational preferences of these molecular balances, while also showing that a simple empirical solvation model can partially account for the attenuating influence of the surrounding solvent.Hunter's α/β hydrogen‐bond model has found use in accounting for the influence of solvents on the conformational preferences of molecular balances.N‐methylation of substituted pyridines with methyl iodide as a model reaction .Having examined the transferability of the through‐space substituent effects in different solvents, we next sought to determine the significance of such effects on reaction kinetics. Inspired by previous work,n Figure  B. Such 2‐X mirrored several important trends observed in the molecular balance series 1‐X:The through‐space substituent effects on the chemical reactivity of compound series 2=0.85, Tables S14, S15). The most reactive control compound was 2‐OMe (50 % completion in ≈35 minutes), while 2‐CN was the least reactive (40+hours to 50 % completion).The experimentally determined rate constants correlate with electrostatic potentials as the X‐substituent was varied .The dimethoxyphenyl derivative ortho‐nitrophenyl compound 2‐f  exhibited similar reactivity to pyridine .The electronically neutral dimethyl‐ and diethylphenyl derivatives  was found to correlate strongly with electrostatic potential of the pyridine nitrogen atom (ESPN) . Despite the numerous commonalities outlined above, the −log10(KX/KH) values determined using molecular balances correlated surprisingly poorly with the −log10(kX/kH) pyridine rate data when both sets of data were determined in [D6]acetone  values determined in six of the eleven solvents examined were found to correlate more strongly with −log10(kX/kH)  values compiled in Table These common patterns are consistent with some transferability of through‐space substituent effects upon varying the X‐substituent. Supporting this assertion, the calculated electrostatic potentials taken over the ined in Dacetone  may also prove useful for benchmarking emerging theoretical solvation models.In conclusion, we have used synthetic molecular balances and a simple model reaction to quantify the importance of through‐space substituent effects on non‐covalent interactions and reaction kinetics, respectively Figure . Experimp Figure  C, left.p Figure . As antip Figure  and 5. Ns Figure . Even thThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The first wave of the Covid-19 pandemic hit Israel in late February 2020. The present study examines patterns of the first wave of Covid-19 morbidity in Israel at the macro level, during the period of late February to early June 2020, when the first wave has faded out. The analysis focuses on the significance of four sociodemographic variables: socioeconomic status, population density, rate of elderly population and minority status (Jewish / Arab identity) of the population in cities with 5000 residents or more. Additionally, we take a closer look into the association between morbidity rates and one SES component – home Internet access.The article is a cross sectional study of morbidity rates, investigated on a residential community basis. Following the descriptive statistics, we move on to present multivariate analysis to explore associations between these variables and Covid-19 morbidity in Israel.Both the descriptive statistics and regressions show morbidity rates to be positively associated with population density. Socioeconomic status as well as the size of elderly population were both significantly related to morbidity, but only in Jewish communities. Interestingly, the association was inverse in both cases. i.e., the higher the SES the lower the morbidity and the larger the elderly population, the lower the community’s morbidity. Another interesting result is that overall, morbidity rates in Jewish cities were consistently higher than in Arab communities.We attribute the low morbidity rates in communities with relatively small elderly populations to the exceptionally high fertility rates in ultra-orthodox communities that sustained increased rates of morbidity; the lower morbidity in Arab communities is attributed to several factors, including the spatial Jewish-Arab segregation. Israel’s first case of Covid-19 was diagnosed on February 26, 2020. On March 14, 2020, 200 cases were confirmed and additional 40–60 cases were being diagnosed daily. Two days later, on March 16, a general lockdown was declared. Mask wearing in public became obligatory on April 12th, but the requirement was hardly enforced for long weeks thereafter. Professionals offered polarized forecasts, ranging from a total of 10 deaths throughout the epidemic, to ten thousand deaths, accompanied by fatal shortage of ICU facilities, ventilators and medical staff. The first wave climaxed in early April with nearly 10,000 active cases and 500–700 additional confirmed cases a day. Since then, the spread started to slow down. Starting April 19, lockdown restrictions were gradually relaxed. As of early June 2020, Israel had roughly 2000 active cases, less than 300 deaths and a fatality rate of 1.67%, substantially lower than that of most West European countries and well below the world average of 6.15%.Scholars have suggested numerous variables that may affect the spread and scope of the pandemic, including medical resources, health coverage, life expectancy, employment rates, income inequality, levels of social connectedness and education, alongside religious affiliation, household size, population age composition, population density and ecological footprint . Of thisSocioeconomic status has often been mentioned in conjunction with rates of Covid-19 morbidity. In the UK, for instance, the disease appears to have spread along class lines. In the early stages of the outbreak, regions with higher SES were over-represented among the sick. Explanations attributed the excess to more frequent traveling and social contacts, alongside better access to testing ),Against this backdrop and the local discourse about the association between Covid-19 morbidity and the four scrutinized variables, we set off to explore the actual findings.This is a cross-sectional study, investigating all of Israel’s residential communities with more than 5000 people . It focuses on Israel’s cumulative Covid-19 morbidity figures since the beginning of national registration in February 23, 2020, up until June 2, 2020, when the first wave of the epidemic was considered to have been contained.Clalit Health Services, Israel’s largest HMO, which presented daily updates of Ministry of Health cumulative morbidity figures, by residential community.  The data includes approximately 197 municipalities in Israel.Data was retrieved from the official publication of Population density in every community, calculated as the number of residents divided by the community’s area of jurisdiction. As such, this is a continuous variable.3The communities’ SES, on a 1–10 (the highest) scale as defined by Israel’s Central Bureau of Statistics. This is calculated as continuous variable.4Proportion of local population aged 65+, thereafter called ‘elderly population’, calculated as the number of 65+ local residents divided by the community’s total population. This is a continuous variable.5Ethnic Nationality as a dichotomous variable, defining ‘Jewish’ and ‘Arab’ communities. This is a dichotomous variable.To supplement this data, we retrieved, for each community, several figures from Israel’s Central Bureau of Statistics’ publications. The added figures included:The dependent variable was the number of confirmed Covid-19 cases in each community in June 2, 2020, and their rate per thousand residents. This continuous variable may be challenged as it heavily depends on the testing policy at any given day. While acknowledging this limitation, we nonetheless consider the official morbidity rate a foundational indicator of local disease spread for several reasons. Morbidity rate was a major component in official assessments of the local spread of the epidemic and as such, was formative of related state policy. This centrality was enhanced by a report, published at the height of the epidemic first wave by the influential Advisory Team to the Center for National Security, that contended, on the basis of emerging local and global evidence, that the number of asymptomatically ill persons was not very large. In light of this clinical and policy shaping significance, we consider the Ministry of Health’s daily morbidity figures a valuable indicator for tracking the spread of Coronavirus in Israel.‘marginal effect’ of each of the study’s variables on local morbidity rates.Data is presented in two stages. First, descriptive statistics outline the distribution of the research variables and the average figures. In the second section, a multivariate analysis is carried out, in order to assess the association between each variable and a community’s Covid-19 morbidity. The regression data is then used to estimate the Looking at Israel’s morbidity rates by SES Fig. , we obseBnei Brak, Elad, Kiryat Yearim, Emanuel, Rechasim in rank 2 and Beit Shemesh, and Zefat in rank 3), which have indeed suffered the highest morbidity in the country. SES level 1 is also populated by orthodox communities. However, it also includes numerous peripheral Arab Bedouin communities. These communities were relatively less affected than communities in the two subsequent levels, as illustrated in Fig. Notably, the SES levels 2 and 3 sustained the highest morbidity rates in Israel. These categories include numerous Jewish orthodox communities  but a mere 8.9% of elderly residents. Jerusalem and Haifa, in contrast, with evidently larger proportions of elderly residents (12.3 and 27% respectively), had strikingly lower morbidity rates: 4.16 and 0.61 per 1000 respectively. A more systematic look into the communities with the highest morbidity rates  of the inter-community morbidity rate variance in Israel at the community level, by SES, density, elderly population and minority status. As shown, SES turned out to be statistically insignificant, but population density, size of elderly population and minority status emerged as significantly associated with morbidity rates. The association of each variable with COVID-19 communities’ morbidity rates is presented in Fig. The regression model thus predicts 38.1% , namely, 2.4 additional patients per 100,000 persons. As for the percentage of a community’s elderly residents, as suggested by the descriptive statistics, the regression shows a significant inverse association between this variable and communities’ morbidity rates, i.e., the smaller the elderly population, the higher the local rate of morbidity (b = − 9.16). In terms of minority status, despite their lower SES and minority status, Arab communities sustained lower morbidity rates, i.e., Jewish communities suffered 1.68 higher morbidity rates compared to Arab communities . Moreover, the Jewish-Arab gap in morbidity proved persistent. Figures The nearly horizontal SES line visualizes the lack of statistically significant association between this variable and the rate of morbidity in Israel. Population density, in contradistinction, emerged as significant, affirming the public discourse that linked crowdedness with increased morbidity. Indeed, population density emerged as the strongest association with communities’ morbidity rates (β = 0.439). A 100 person increase per kmb = − 9.1. In termThe calculation is based on the regression model, assuming that all other variables are held constant. The figures demonstrate that the higher the population density, the higher the community’s morbidity rates and the larger the elderly population, the lower the morbidity rate. Equally important: at any given value of population density or size of elderly population, the gap in morbidity rate between the Jewish and Arab cities persists, with morbidity rates consistently lower in Arab communities.In the light of the emergent gap in morbidity between Arab and Jewish communities, we divided our calculation and produced the average values of the scrutinized sociodemographic variables alongside Covid-19 morbidity rates in Jewish vs. Arab communities. Table The table reveals a clear Jewish-Arab difference: with communities twice or more richer, denser and older, Israel’s Jewish cities sustained 2.6 times the morbidity of their Arab counterparts. Whereas these figures accord with international findings regarding population density and proportion of elderly residents, the higher morbidity in richer cities is exceptional. In other words, Israel’s Arab communities, despite being much poorer and despite belonging to an underprivileged minority, sustained less than half the morbidity of their wealthier Jewish neighbors. Therefore, we examined how these variables are associated with morbidity in Jewish vs. Arab communities. The linear regression analysis presented in Table The analysis shows that when considered on its own, SES (Model 1) becomes significant in the Jewish communities though not in the Arab ones. Thus, in Israel’s Jewish communities SES was associated with morbidity in a manner similar to that observed elsewhere, i.e., poorer communities were more vulnerable to the virus than wealthier ones. More concretely, a rise in one SES rank in a Jewish community was associated with a reduction of 0.373 sick person per 1000. In the Arab sector, no such association was found. However, once the other variables are added (Model 2), the association of SES with morbidity once again becomes statistically insignificant also in the Jewish sector. Population density (Model 2), however, proved significant and equally influential in both sectors.The third variable, proportion of elderly population, also emerged as statistically insignificant in the Arab communities but significant and inversely associated with morbidity in Jewish communities, i.e., a smaller elderly population was associated with higher morbidity. Another substantial inter-sectorial difference related to the explained variance. While explaining nearly 38% of the variance in morbidity rates in Jewish communities, Model 2 accounts for less than 11% in Arab settlements. The model thus reaffirmed the lay perception that population density was positively associated with morbidity rates. However, in contrast to international findings, SES was statistically insignificant to morbidity rates in the Arab communities and even in the Jewish sector, the association was weak.Finally, some Israeli commentators attributed the high morbidity rates in some ultra-orthodox communities to the scarcity of home and mobile Internet access in these communities, that wish to maintain but minimal contact with their non-orthodox surrounding.  This virtual detachment was assumed to underlie the communities’ lack of awareness and belated application of the protective measures and lockdown guidelines. We therefore zoomed in on this specific component of the SES index and conducted a specific linear regression.The general regression revealed no association with internet access. Sector specific analyses Table  found thHowever, the latter association was substantial: a 10% increase in home internet access in Jewish communities was associated with a morbidity decline of 0.3 person per 1000. The resemblance to SES patterns is reasonable, given the fact that internet access is a component of the SES index.Figure We now turn to discuss the observed trends and particularities and propose some lines of explanation.opulation density was the only variable that emerged as statistically significant to morbidity in the entire Israeli population. In Arab communities, the lower density - 2037 vs. 3853 persons per km2 – might have contributed to the lower morbidity. The association of SES with communities’ morbidity solely in Jewish cities probably reflects the relatively narrow range of SES distribution of Israel’s Arab communities, which are largely clustered at the lower end of the SES scale. However, in the Jewish sector, too, SES was significant only when analyzed on its own and then, too, the association was moderate: massive differences in morbidity were observed among communities with similar SES7The current analysis revealed increased morbidity rates in dense, low SES Jewish communities with small elderly populations and low access to household Internet. This description befits first and foremost Jewish Orthodox communities. As mentioned, these communities have indeed suffered the highest morbidity rates in Israel. More generally, pelderly population is probably the most peculiar finding of the present study. Israel’s population is much younger than that of other industrialized countries. The main reason for the gap is the local fertility rate of 3.11 children per woman, which is nearly twice the OECD average of 1.7.Ibe. BeyondBeyond this particular difference, the analysis revealed that when density level, SES or size of elderly population are held constant as average, the prediction of morbidity rate in the Jewish communities is 1.68 times higher than in Israel’s Arab communities. This lower morbidity rate among the country’s poor minority population, as compared to the wealthier majority group, counters findings from other countries. Moreover, multi-generation households, another variable associated with higher morbidity, as mentioned, are more prevalent in Israel’s Arab communities than in Jewish ones ) As of May 20, 2020, there were a total of 1040 confirmed Covid-19 cases and five deaths in Arab communities in Israel, since the outbreak of the epidemic .To sum up, Arab communities in Israel were relatively less affected by the Covid-19 epidemic than Jewish communities, despite their relative poverty, minority status and prevalent multi-generational households. Lower population density and smaller families, alongside highly efficient community self-organization and collaboration with state bodies resulted in exceptionally low morbidity and mortality rates. In contradistinction, high morbidity was observed in Israel’s poor, crowded Orthodox communities, especially ones with very small elderly populations.The observed trends and differences may possibly offer an insight about potential state measures and the protection and inclusion that they have offered to marginalized communities in the context of pandemics. A comparison with morbidity patterns during Israel’s second wave of Covid-19 morbidity may reveal how different containment measures have affected the health and lives of various subpopulations across the social ladder.The first limitation of the current study is the focus on confirmed cases. As noted, this figure closely depends on the prevalence of testing. Moreover, assessments of the proportion of asymptomatic ill people vary widely from eight to 54% . Additi


Microbial communities underpin the Earth's biological and geochemical processes, but their complexity hampers understanding. Motivated by the challenge of diversity and the need to forge ways of capturing dynamical behaviour connecting genes to function, biologically independent experimental communities comprising hundreds of microbial genera were established from garden compost and propagated on nitrogen-limited minimal medium with cellulose (paper) as sole carbon source. After 1 year of bi-weekly transfer, communities retained hundreds of genera. To connect genes to function, we used a simple experimental manipulation that involved the periodic collection of selfish genetic elements (SGEs) from separate communities, followed by pooling and redistribution across communities. The treatment was predicted to promote amplification and dissemination of SGEs and thus horizontal gene transfer. Confirmation came from comparative metagenomics, which showed the substantive movement of ecologically significant genes whose dynamic across space and time could be followed. Enrichment of genes implicated in nitrogen metabolism, and particularly ammonification, prompted biochemical assays that revealed a measurable impact on community function. Our simple experimental strategy offers a conceptually new approach for unravelling dynamical processes affecting microbial community function.This article is part of the theme issue ‘Conceptual challenges in microbial community ecology’. ApproprNew strategies for investigation are required that are process-focused . IdeallyEqually desirable are approaches that provide a link between genes, their spatial and temporal dynamics, and community function, achieved using strategies that leave as far as possible the natural complexity of communities undisturbed. Here we show—in a proof-of-principle experiment—that this can be achieved via an experimental strategy that combines theory governing the behaviour of selfish genetic elements –23 (SGEs2.(a)3 compost heap and each placed in one of ten 140 ml bottles (mesocosms) containing 20 ml nitrogen-limited minimal M9 medium plus cellulose (a 4 cm2 piece of paper) as sole carbon source. Mesocosms were incubated on a laboratory bench without shaking and with lids left unsealed. After a two-week period, the initial piece of paper from each mesocosm was separately transferred to a new bottle containing fresh M9 medium plus a new piece of paper. Following a further two-week incubation period, we considered the communities to have adjusted to laboratory conditions. At this time point, mesocosms were vortex-mixed and 1 ml of cellulosic slurry from each bottle was transferred to two new mesocosms containing 19 ml fresh M9 medium plus paper giving a total of 20 paired communities. This transfer, four weeks after initial establishment, was deemed time point zero (T0). Thereafter and for the ensuing 48 weeks, communities were serially propagated every two weeks (T1 to T24) by transferring 1 ml of slurry to fresh bottles. One set of each pair of bottles was labelled ‘vertical’ ) and the second of each pair labelled ‘horizontal’ ). Details of the treatments and their significance are elaborated below, but at this stage, with a focus solely on the question of diversity in cellulose-based mesocosms, we acknowledge treatment effects by name only.Experimental communities were established from a diverse primary source: garden compost. Ten independent 1 g samples of compost were taken from 10 different regions of a single 1 m0, and 20 T1 and T24 communities and the data interrogated for sequence reads mapping to 16S rDNA . Communities at T0 harboured on average approximately 140 bacterial genera (s.d. ± 23). At T24 the number of genera detected increased in all communities to an average of approximately 200 (s.d. ± 32), indicating an increase in abundance of rare types . Diversity levels were highly similar in both HCs and VCs . Rank abundance distributions from pooled HCs and VCs at T1 and T24 are shown in 24 distribution to be markedly flatter in both cases, consistent with an overall tendency for rare types to have increased during the course of the year-long selection. Rank abundance distributions for each T1 and T24 HC and VC, plus the rank order (and change therein) of the most common genera are shown in the electronic supplementary material, figure S2a,b. Electronic supplementary material, figure S3 is a multidimensional scaling plot showing that communities at the start and end of the experiment differed markedly in genera composition and there was no sign of convergence after 48 weeks. Among the most abundant genera, only two harbour species commonly associated with cellulolytic ability [Cytophaga and Cellvibrio), while others are better known for roles in ammonification [Azospirillum and Paenibacillus (nitrogen fixation); Rhodanobacter and Pseudomonas (dissimilatory nitrate reduction/denitrification); electronic supplementary material, figure S2a,b). Single-celled eukaryotes were also present in all mesocosms with several unexpectedly maintaining populations of nematodes through the 48 week selection period.Diversity through 48 weeks of propagation was addressed by metagenomics: total DNA was extracted from each of the 10 T ability  (Cytophafication  (Azospir(b)An important goal is the development of strategies that link genes to community function by way of the process. An ideal experiment is one that alters a single ecological or evolutionary process and compares the outcome to a set of replicates in which the process is unaffected. For example, evolutionary biologists interested in the evolutionary consequences of sex have taken advantage of yeast in which it is possible to genetically engineer sexual and asexual types that are otherwise isogenic ,30. DiffWe reasoned that a similar manipulation might be possible in the context of microbial communities. Recombination mediated by HGT is loosely analogous to sex, albeit to a form of sex that takes place at the level of communities. Moreover, like genetic manipulations affecting sexual reproduction in yeast, the extent of HGT can be controlled by manipulating the evolutionary fate of SGEs ,23. SuchThe critical manipulation is one that affects the likelihood that SGEs encounter new hosts. Frequent exposure to new hosts drives dissemination and evolution of SGEs and linked genes . Conversvertical and horizontal treatment designations referred to above reflect manipulations that are predicted to either restrict, or facilitate, respectively, HGT mediated by SGEs. The vertical regime involved serial transfer of material from mesocosm to mesocosm. SGEs within these communities were denied opportunity to encounter new hosts: transfer of SGEs was vertical and the extent of HGT is expected to be limited 0 community and each of the 20 T1 communities  and sequenced (0 and descendant HCs and VCs (e.g. T0-1 and descendants T1V-1 and T1H-1) DNA sequence data were interrogated to identify reads found solely in (and therefore unique to) the horizontal mesocosm (e.g. T1H-1). Such unique reads are likely to stem from SGEs, or be associated with SGE activity, originating from an allopatric community.To determine whether the horizontal regime promoted the movement of SGEs, total DNA was extracted from each TAnalysis of the sequences unique to each horizontal community resulted in identification of approximately 26 million unique reads from a total of approximately 152 million reads across all 10 communities, with an average of approximately 2.6 million unique reads per community . Assembly of reads into contigs revealed an average of 3352 contigs per HC greater than 1 kb in length. The mean maximum contig size per assembly was 82 kb .1H-1_35969) containing numerous genes characteristic of phages is shown in the electronic supplementary material, figure S4a.After extracting open reading frames (ORFs), interrogation of the Conserved Domain Database (CDD)  showed 11H-1_35969 and 19 other representative phage-like entities  across independent mesocosms was determined by mapping total reads from each horizontal and vertical metagenome onto each of the 20 phage-like contigs . Within two weeks, this contig had amplified and spread to HC 1, 5 and 6 . These include genes implicated in cellulose degradation, defence against SGE invasion, nitrogen metabolism and siderophore biosynthesis  –38. Part(d)0 communities into VCs and HCs, we complemented existing DNA sequence from T0 and T1 and T24 with DNA extracted from all HCs and VCs at time points T2, T3, T4, T10, T16 and T20 yielding a total of 180 metagenomes . These 180 metagenomic datasets ) spread across 48 weeks allowed the abundance and distribution of SGEs and associated genes to be determined by read mapping to contigs assembled from T0 data. The spatial and temporal dynamics of two phage-like contigs and two contigs encoding β-glucosidases are shown in figure 4a–d.Given evidence indicative of the amplification and dissemination of DNA via the horizontal treatment just two weeks after splitting T1H7_13351 (a), this phage-like element was detected only in community C4 at T0, but was probably present also in communities C1 and C2, although below the limit of detection. By week 3, the phage-like element was present in all HCs. In VCs, the element persisted in community C4 for 22 weeks and transiently in C1 and C2, but was present at 48 weeks in all but one HC. Within HCs, a pattern observed on four occasions saw the phage-like element go extinct , but re-emerge and persist at latter time points.Focusing on PhageContigTH7_13351 a, this p(e)0 and all T24 HCs and VCs . For the ensuing analysis, we have avoided standard statistical testing because horizontal microcosms, by virtue of the movement of SGEs and linked genes among communities, cannot be treated as independent entities. Nonetheless, trends are clearly evident from the data displayed in 0 community—median value, interquartile range and full data range.To determine whether the horizontal transfer regime affected the abundance and distribution of functional genes, the MG-RAST database was interrogated with sequence reads from all TThe relative abundance of reads assigned to 13 of 28 functional categories at subsystems level 1 , includia). Changes were observed in the relative abundance of reads mapping to genes involved in both ammonia assimilation and dissimilation (ammonification). Such changes are consistent with the nature of the selective environment. This prompted a query of the extent to which these changes may have been influenced by the movement of genes via SGEs.The nitrogen-limited nature of the culture medium, combined with evidence of selection favouring genes involved in nitrogen metabolism, led to closer focus on this essential resource. The MG-RAST database was again interrogated, but this time at subsystems level 2 , which pfigure 524 metagenomes. T24 HC metagenomes were compared to their paired T24 VC metagenomes as well as to all metagenomes from VCs from earlier time points . This showed a greater number of unique sequences in the HCs, but also showed a fraction of unique sequences present in VCs. The latter is to be expected given that T0 communities must contain rare sequences that through the course of the selection experiment became common—and is consistent with data on changes in abundance of genera . Nonetheless, the ‘deep’ T0 metagenomes were used in all subsequent analyses.A modified version of the bioinformatic pipeline developed to identify sequences unique to HCs (Methods) was applied to the Tf genera . To see b; electronic supplementary material, table S9).To determine the fraction of unique reads that mapped to genes involved in nitrogen metabolism, the final sets of unique reads were obtained from HCs and VCs; they were assembled, ORFs identified and functionally categorized using the CDD. HCs contained more unique ORFs predicted to be involved in nitrogen metabolism compared to VCs . HCs were enriched in several functional classes of gene, especially those with predicted roles in regulation and ammonification  the concentration of nitrate, nitrite and ammonia were determined in the Tb; electronic supplementary material, table S11). A comparison between T0 communities and T24 VCs showed no change  supporting the conclusion that movement of ecologically significant genes via SGEs was responsible for the functional difference between HC and VC.To check the robustness of this finding, the concentration of ammonia was measured during the course of a two-week period. Despite substantive variability in the composition of the independent communities , a significantly greater concentration of ammonia was detected in HCs at each of nine sampling occasions throughout the two-week period  genotypes are propagated under defined laboratory conditions with variation arising solely by de novo mutation and where selection is the primary arbiter of mutation success ,25. ThisWe sought to apply the same kind of simplifying experimental approach to the study of microbial communities, however, doing so is not straightforward. Simply substituting a community of microbes in place of a single bacterial genotype, followed by recapitulation of a standard serial transfer experiment, would not be analogous to propagating a single bacterial genotype as done, for example, in the work of Lenski . In the If it makes no sense to assume that communities adapt in a manner analogous to single bacterial populations, then attention shifts away from the focus on mutation and selection affecting individual units, and turns to possibilities that might stem from manipulation of evolutionary processes such as recombination, or migration that could conceivably have community-level effects. We chose to manipulate the former, seeing the possibility to promote, or retard HGT, via a simple mixing regime predicted to affect the evolutionary fate of SGEs, which in turn, might connect genes to community function.in vitro environment not too dissimilar from that encountered in nature. Additionally, component species can in principle be isolated and cultured on laboratory agar with glucose as sole carbon source (glucose being the utilizable metabolic break-down product of cellulose). However, the primary consideration for use of cellulose was that it is difficult to degrade [r-selected types. In effect, while the means and mode of propagation involved serial transfer, communities probably experienced culture conditions more reminiscent of those provided in chemostat culture.Our focal communities were established from a highly diverse environment (garden compost) and propagated on cellulose as sole carbon source. The decision to use cellulose was in part a nod to simplification, but was also motivated by the fact that garden compost is rich in this material, thus ensuring an  degrade , thus alLevels of diversity recorded from analysis of metagenomic data were remarkably high, remaining stable—and even increasing—through the course of the 48 week period of propagation . This isAlthough noted only in passing, and from examination of the mapping of DNA sequence reads to genes connected with nutrient cycling (data not shown), cycles for replenishing all major nutrients appeared to have been established within each mesocosm, with that involving nitrogen being most evident. Each community harboured a single dominant free-living nitrogen-fixing bacterium, but the identity—even at the genus level—differed between mesocosms. Attempts to establish such complex communities by choosing key players before the fact, would probably fail. That such diversity and stability can arise from a single gram of compost speaks to an abundance of redundancy in these natural systems ,53.Given the experimental design with vertical and horizontal treatment regimes, it is natural to query the relationship between treatment effects and patterns of diversity. At the level of number of bacterial genera and shape of rank abundance curves, no difference was detected and in fact communities showed remarkably similar overall trends. However, differences are apparent when focusing on the rank of individual genera. These are evident in the butterfly plots in the electronic supplementary material, figure S2b and show difference both between independent communities but also differences between vertical and horizontal treatments within communities. Because our experimental design did not include technical replicates  no statistical analysis is possible. Nonetheless, notable is the similarity of changes in genus rankings between vertical and horizontal treatments from the same communities. Also notable is the fact that there was no evidence of convergence in diversity in the horizontal treatment after 48 weeks . Although unsurprising, this emphasizes the distinctness of the communities despite being connected via movement of SGEs and linked genes.The pooling of material from each mesocosm that passed through a 0.2 µm filter to produce an SGE-cocktail, followed by its mixing and regular distribution among HCs, was evidently effective in mobilizing SGEs and promoting horizontal transfer of genes of ecological significance. In fact, the magnitude of the effect, combined with capacity to detect it via identification of DNA sequence reads unique to each horizontal community, exceeded expectation. Within just two weeks of implementation, approximately 17% of DNA sequence reads within HCs were derived from allopatric communities. That these reads could be assembled into contigs containing genes of ecological relevance, including those involved in cellulose degradation, emphasizes the dynamism of processes driven by SGEs.One consequence of this dynamism is the application of the protocol to detect—at the community level—traits under selection. The suite of unique DNA sequences amplified and disseminated across communities suggests that these genes matter. In the context of conditions experienced by the compost-derived communities, this makes sense. Community members are expected to have experienced selection for the ability to degrade cellulose: this is indicated by the abundance of horizontally transferred genes predicted to be involved cellulose degradation . Similarly, as mesocosms were nitrogen-limited, selection was expected to favour the movement of genes involved in nitrogen metabolism. That this happened is indicated by the prevalence of genes implicated in nitrogen metabolism among the set of DNA sequence reads unique to HCs. Not appreciated at the outset was that communities, according to the signature acquired from unique sequence reads, were iron limited. With hindsight, this was to be expected. That genes encoding CRISPR are also enriched is especially significant and suggests an unappreciated dynamic between hosts, the defence systems maintained to protect bacterial hosts against phages, and benefits that accrue from the horizontal dissemination of these systems .Without further experimentation, the identity of the elements responsible for horizontal spread of genes is unknown, and so too is the relationship between the genes amplified and disseminated across mesocosms, and the vehicles for their dissemination. While some assembled contigs show features typical of phages, many do not. This may be a consequence of working with metagenomic data, but may also reflect the existence of elements whose nature and dynamic remains to be discovered.One of the larger assembled contigs shown in the electronic supplementary material, figure S4b is a case in point: at approximately 80 kb, hints as to its identity are expected, and yet while carrying genes predicted to encode glycoside hydrolyses—implicated in cellulose degradation—the only suggestion of ability to be both mobilized and disseminated is via linkage to a single integrase. Acquisition of long-read sequence data may aid understanding, but it is also possible that the captured dynamic—the rapid amplification and dissemination of ecologically significant genes—is attributable to ‘fortunate accidents’ mediated by, for example, lytic phages or other elements that happen on rare occasion to capture genes of utility to hosts, with the feedback between genes that prove useful to hosts and the amplifying effects of selection driving their dynamic. This points to the possibility of processes that unfold at the level of diverse communities that remain to be understood.1H7_13351 (a) raise numerous questions. Extinction within VC4, but persistence in all HCs at 48 weeks (with the exception of H1) raises questions as to the causes. Using recently developed Hi-C [Irrespective of the nature of the disseminating vehicles, the experimental approach outlined allows the dynamics of elements amplified and disseminated to be tracked. Examples are shown in figures H7_13351 a raise nped Hi-C ,19,55 anped Hi-C , it ough0 communities) in VCs . Additionally, increased ammonia in the HCs is consistent with the prediction that the horizontal movement of DNA will increase the rate at which community function improves.The association established between genes involved in nitrogen metabolism and enhanced activity of SGEs in HCs is correlative, but is nonetheless linked to horizontally transferred sequences that map to genes with predicted roles in ammonification. The association is further linked to data that demonstrate a significant effect of the experimental treatment on concentrations of ammonia in HCs , but no The two processes that generate ammonia: nitrogen fixation and nitrate ammonification, both require environments devoid of oxygen (or mostly so in the case of nitrate ammonification) ,56. It iThe genomic era has done much to resolve the controversy surrounding the importance of HGT as a driver of evolutionary change –60. DireMicrobial communities are dynamical systems defined by complex ecological interactions and evolutionary feedbacks . The cha4.(a)2 piece of cellulose paper (Whatman cellulose filter paper). All incubations were performed in 140 ml sterile glass bottles with loosened screw caps allowing for gas exchange between the community and the environment. Incubation was at room temperature without shaking. Compost was sampled from the Square Theodore-Monod compost heap  in February 2016. During the primary incubation period of two weeks, the piece of cellulose was suspended by a wire from the screw cap into the media allowing for paper colonization. After two weeks, the community was transferred by moving the wire-suspended cellulose paper into 20 ml of fresh M9 minimal media that also contained a new 4 cm2 piece of cellulose paper in suspension. Two more weeks of incubation provided the opportunity for colonization of the new piece of suspended cellulose paper thus eliminating the need for a wire-suspended community for each transfer. All community transfers were performed by vortexing each bottle at maximum speed until the cellulose fibres were disaggregated into a slurry.Microbial communities were initially established by placing 10 independent 1 g samples of fresh compost into 20 ml of M9 minimal media (the nitrogen source was ammonium chloride (0.935 mM)) supplemented with a 4 cm(b)g for 10 min and the pellet was stored at −80°C for later processing. DNA was extracted from the founding T0 communities  as well as transfers 1, 2, 3, 4, 10, 16, 20, 24  using the soil DNA extraction kit (Norgen Biotek). DNA sequencing was performed using the MiSeq and NextSeq platforms for all time points and T0 communities with additional deep sequencing of the T0 communities using the HiSeq platform.Vertical and horizontal transfers were performed at exactly the same time every two weeks. Before each transfer aliquots of the community were taken for glycerol stocks, DNA extraction and phage storage. Glycerol stocks were created by mixing 500 µl of cellulose-microbial slurry with 500 µl of 80% glycerol and stored at −80°C. For DNA extractions 2 ml of cellulose-microbial slurry was centrifuged at 13 000n = 10) followed by centrifugation and passage through a 0.2 µm filter to remove microbial cells. Recognizing the possibility that ultra micro-bacteria might escape sedimentation and pass through the filter, metagenomic sequence reads were recruited to seven available reference genomes. A maximum of 0.018% of reads across all horizontal and vertical mesocosms at 48 weeks matched these reference genomes .Transfers at two-week intervals were performed as described in (c)rinSeq [−5, minimum alignment length of 100 base pairs, and a minimum percentage identity of 90%. ‘Unique’ horizontal and vertical sequences were assembled using MEGAHIT [r-Hit [0 metagenome  and the paired vertical metagenome using BLASTn. Horizontal-T0 and horizontal–vertical sequence matches were removed, thus leaving a set of unique horizontal sequences that could not be identified in neither the founding T0-metagenome, nor the paired vertical metagenome. Sequence matches were determined using a minimum e-value threshold of 1 × 10−5, minimum alignment length of 100 base pairs, and a minimum per cent identity of 90%. The unique horizontal sequences were then assembled, ORFs were extracted, and predicted protein domains were determined through comparison to the CDD. To determine the impact of increasing the sequencing depth of the T0 communities on the final percentage of unique sequences, T0 metagenomes were sequenced using the HiSeq platform and reads from each mesocosm split arbitrarily into eight metagenomes each consisting of about approximately 40 million sequences. The unique analysis pipeline was then applied using the first arbitrary T0 metagenome and any matching sequences were removed. The process was repeated with all eight T0 arbitrarily defined metagenomes thus revealing the extent to which additional sequencing affected the ability to detect rare sequences.DNA sequences were demultiplexed using bcl2fastq, paired ends were joined using FLASh  and initrinSeq  with a mrinSeq . SequencrinSeq  with a m MEGAHIT , ORFs wi MEGAHIT , compare MEGAHIT  using RPT [r-Hit . Sequenc(d)24) and T0 were re-established by placing 100 µl of glycerol stock into 20 ml of M9 minimal media supplemented with a 4 cm2 piece of cellulose paper and incubated for two weeks. One millilitre of the cellulose-community slurry was transferred to 19 ml of M9 minimal media with a fresh piece of cellulose and incubated for an additional two weeks. One millilitre of the community was then transferred to a new bottle and 100 µl of surrounding media was sampled at various time points during the two-week incubation period to determine ammonia/ammonium, nitrate and nitrite concentrations. The concentrations of all three nitrogen species were determined using fluorometric assay kits according to the manufacturer's protocol (Ammonia Assay Kit, Sigma-Aldrich, and nitrate/nitrite fluorometric assay kit; Vertical and HC from the 1-year time point (24 transfers, T


Cupiennius salei); however, much less is known about the distribution and function of chemosensory organs. Furthermore, our knowledge on how the sense organ pattern develops on the spider appendages is limited. Here we analyse the development of the pattern and distribution of six different external mechano- and chemosensory organs in all postembryonic stages and in adult male and female spiders of the species Parasteatoda tepidariorum. We show that except for small mechanosensory setae, external sense organs appear in fixed positions on the pedipalps and first walking legs, arranged in longitudinal rows along the proximal-distal axis or in invariable positions relative to morphological landmarks . A comparison to other Entelegynae spiders shows that these features are conserved. We hope that this study lays the foundation for future molecular analysis to address the question how this conserved pattern is generated.Spiders are equipped with a large number of innervated cuticular specializations, which respond to various sensory stimuli. The physiological function of mechanosensory organs has been analysed in great detail in some model spider species (e.g. The online version of this article (10.1007/s00427-020-00655-8) contains supplementary material, which is available to authorized users. Arthropods have a sophisticated sensory system which mediates environmental interactions and supports essential activities such as foraging and mating Stevens . BesidesParasteatoda tepidariorum, with specific focus on the chemosensory (CS) setae, which are understudied in spiders. Previous publications suggest that CS setae are more abundant on the anterior appendages  on the appendages of the spider s Foelix  and we ts Foelix . The peds Foelix .In arthropods, ESOs originate from precursor cells in the epidermis, which form cuticular specializations such as bristles, sockets and slits, in addition to the neural part of the sense organ , or sturdy (bristle-like). Many exhibit a serrated surface Table  but smooThe tarsal organ is an open pit located on the dorsal tarsus. It contains several sensilla with single pores at the tip suggesting an olfactory function but electrophysiological recordings also indicate responses to humidity and thermal stimuli Barth . The CS P. tepidariorum in all postembryonic and adult stages and both sexes. The aim of the paper is to contribute to our understanding of the evolution of sense organ patterns and to provide the morphological basis for future molecular genetic studies in this model spider species.Here, we describe the distribution of these 6 types of sensilla in the pedipalps and L1 of P. tepidariorum shows a dome-like shape and a central pit  setae are present on the distal most 2–4 podomeres of the pedipalp and L1, respectively, in addition to the pedipalp coxa Fig. , followeOn the tibia of L1, the first couple of CS setae appear in fixed positions on the dorso-posterior side close to the metatarsal joint (2nd instar) and on the dorso-anterior side in the middle of the podomere between the landmark MS setae 3rd instar) Fig. . In the rd instarIn the 1st instar, most of the tarsal CS setae are located at the distal tip Fig. . The firP. tepidariorum, the CS setae of the walking leg are mainly found on the distal podomeres  in all Entelegynae spiders analysed , has three CS rows on each side of the leg in addition to a dorsal and a ventral row . In contrast to other spider species, the trichobothria are not clustered in P. tepidariorum, rather, they are aligned in an offset dorsal row   Barth .Slit sensilla are present from the 1st larval stage onward, initially only in the distal podomeres . Most slit sensilla cannot be identified individually throughout development due to a lack of landmarks and intercalary growth of the appendages. However, two slit sensilla located at the distal tip of the tarsus are visible in all postembryonic stages and in the adult, both in L1 and the pedipalps Figs. b. The slP. tepidariorum is similar to the pattern in C. salei and A. bruennichi  Fig. b. There C. salei and A. bruennichi  and in L1  of P. tepidariorum, considering the different sizes of the appendage joints. In all three spider species, there is a single lyriform organ on the coxa and on the metatarsus of L1 but the numbers vary in the remaining joints. A. bruennichi and P. tepidariorum also show sex-specific differences in the number of lyriform organs on the tibia of the pedipalps. While there is a lyriform organ at the distal tibial joint in A. bruennichi males, the sense organ is missing in females. The reverse is the case in P. tepidariorum.Compared to P. tepidariorum compared to other Entelegynae spider species from which data are available  in late postembryonic and adult stages, and cannot be individually identified, except for a few large bristle-like setae. In contrast, all other ESOs seem to appear in fixed positions. CS setae, slit sensilla and trichobothria are arranged in longitudinal rows along the proximal-distal axis. In addition, there is a dense field of CS setae at the tip of the pedipalpal and L1 tarsi and a cluster of slit sense organs on the pedipalpal coxa and L1 trochanter  with lactic acid (1:1 lactic acid to Hoyer’s medium) was used as a mounting solution clearing the cuticles of soft tissues and pigments. The mounted samples were placed into an oven at 65 °C overnight. An inverse Leica microscope (DM IL) and corresponding LAS software (version 2.8.1) were used for image acquisition of cuticle preparations.For the analysis of the external sense organs, we used either the exuviae, which were preferred because of their transparency, or whole mounts of the spider appendages. Leg and pedipalp preparations of spiders were obtained by placing them for a few minutes at − 20 °C to anaesthetize them. Using forceps and a stereomicroscope, pedipalps and 1st walking legs were removed. The specimens were mounted between two coverslips enabling the imaging from both sides (anterior and posterior). Hoyer’s medium  and the 1st walking legs of adult female spiders (removed from anaesthetized spiders as described above) were fixed in 1:1 heptane and glutaraldehyde/paraformaldehyde (3%) and 0.01% Triton X-100. The detergent reduces the surface tension which enables the samples to submerge. The samples were fixed overnight at 4 °C and dehydrated using an ascending alcohol series . Samples were incubated for 15′ at each concentration and then chemically dried using HMDS (Sigma-Aldrich) by transferring the samples into a glass dish. The samples were washed once with 1:1100% EtOH and HMDS and replaced with 100% HMDS, which was left in the glass dish for evaporation overnight under the fume hood. Dried samples were carefully mounted onto aluminium stubs prepared with sticky tape. For SEM imaging (FEI Quanta 3DFEG and the FEI Inspect F electron microscopes), the samples were gold sputtered (Agar auto sputter coater model 108A).Light microscope images were used to count the 6 different ESOs on each side of the podomeres . For the 1st to 5th instars, we counted the ESOs of 6 appendages each for pedipalps and L1. For the subadult and adult stages, we counted 2 appendages each for pedipalps and L1, and for females and males, respectively.Microsoft Excel  was used to document data, calculate the mean and standard deviation (SD), as well as for creating bar charts. The schematic drawings are based on light microscopic images and were generated with Inkscape (version 0.92.3) and Adobe Illustrator (version 16.0.0).ESM 1(PDF 22718 kb)


Nephrogenic diabetes insipidus (NDI), which can be congenital or acquired, results from the failure of the kidney to respond to the anti-diuretic hormone (ADH). This will lead to excessive water loss from the body in the form of urine. The kidney, therefore, has a crucial role in maintaining water balance and it is vital to restore this function in an artificial kidney. Herein, an ultrasensitive and highly selective aptameric graphene-based field-effect transistor (GFET) sensor for ADH detection was developed by directly immobilizing ADH-specific aptamer on a surface-modified suspended graphene channel. This direct immobilization of aptamer on the graphene surface is an attempt to mimic the functionality of collecting tube  Anti-diuretic hormone (ADH), also known as arginine-vasopressin or neurohypophysial hormone, is an imperative peptide for maintaining the plasma osmolality in the human body . TheoretFrom 1950 to the present, several approaches have been developed and implemented for detecting ADH, namely bioassays, immunoassays, radioimmunoassays and enzyme-linked immunoassays (ELISAs). The extensive time consumption, extravagant procedure cost and low specificity of bioassays lead to the development of the immunoassay approach . HoweverCurrently, there are many ADH assay kits available on the market, mostly dominated by ELISA-type kits such as the ADH/ vasopressin (VP)/ arginine-vasopressin (AVP) ELISA Kit from Biomatik. The lowest threshold exhibited by these kits is 0.5 pg/mL . Althougn−1·kg−1 , the limn−1·kg−1 . TherefoOne of the most promising tools to resolve these issues is the biosensor. Among the variety of electrical biosensing architectures reported ,15, deviA graphene-based field-effect transistor (GFET) with suspended graphene (the suspended graphene bridges the source and drain electrodes) holds more advantages than an embedded architecture (graphene supported by silicon dioxide substrate). This is because charge carriers in a suspended graphene FET offer high mobilities up to ded GFET ,20. In aded GFET . Howeverded GFET . This leIn this work, a FET-based biosensor was developed to detect ADH in experimental setting. A suspended graphene-based FET was fabricated for the ultrasensitive, selective and rapid detection of ADH. Moreover, ADH-specific aptamers were immobilized on the graphene channel as an attempt to mimic the functionality of the collecting tube Field-effect transistor (FET) devices were fabricated by using the conventional photolithography process and metal lift-off technique as described previously . Single Two microliters of 1 M of potassium hydroxide (KOH) with pH 9.0 acquired from Qrec (Asia) Sdn. Bhd. was pipetted onto the graphene layer at the channel area. A phosphate-buffered saline ) was used as the washing buffer. The device was characterized using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy to inspect for the successful chemical activation of the graphene layer. The process then proceeded with the functionalization of the graphene layer that comprises of two main stages which are surface modification and immobilization of aptamers.The surface modification began with the introduction of 2% (3-aminopropyl) triethoxysilane (APTES) in 30% ethanol solvent to formulate an amine functionalized surface which was then left for 2 h at ambient condition. PBS buffer was used to rinse and wash the device three times to remove unbounded APTES residues from the surface. Subsequently, 2.5% glutaraldehyde (GA) was applied to the chemically modified graphene surface. This was left for an hour for the molecules to establish covalent bonding with the amine functionalized surface. The surface was then rinsed, washed and left till dry. After that, 1 µM of ADH specific-aptamer with the sequence of 5’ TCACGTGCATGATAGACGGCGAAGCCGTCGAGTTGCTGTGTGCCGATGCACGTGA-3’  , urease, glucose oxidase and human serum without ADH hormone was independently applied at the channel area and electrical measurements were recorded using the Keithley 2400 Sourcemeter SMU Instruments. Similarly, human serum with ADH was tested to check for the specific functioning of the biosensor in human body conditions.The image of the fabricated device obtained from the optical microscope is illustrated in The chemical composition of elements that made up the fabricated GFET was inspected and verified by performing the energy-dispersive X-ray analysis (EDX) on the GFET area comprising the source, drain and transducing channel. The 3D image from the characterization of surface morphology by the atomic force microscope (AFM) is illustrated in Raman spectroscopy was performed to analyze the structural and electronic properties of the graphene before and after transferring onto the device . A 532 nectively . The ratBased on the data obtained and tabulated in 350 cm−1 c. This pl doping ,29. The image of the FET after graphene transfer is shown in graphene . HoweverIt is also worth mentioning that there is a sharp peak at 2450 orted by  in 2005.orted by ,33,34. TThe ATR-FTIR transmission spectra of the graphene layer for four consecutive surface functionalization processes that begins with chemical activation a, surfacFour new peaks emerged after the graphene layer was activated with 1 M of KOH in comparison with bare graphene a. A peakgraphene . Valanced out by . The pea bending  and out- bending . Therefo bending . The KOH-activated graphene was then modified with APTES. From Subsequently, 2 µL of GA was added on the transducing layer. GA is a homo-bifunctional cross-linker with aldehyde groups on both ends with carbon chain spacer . The alds report . Peaks aIn the last stage, ADH-specific aptamer which is the bioreceptor for the ADH biosensor was immobilized on the surface-modified graphene. The measurement was taken only after 15 min to ensure the complete immobilization of ADH-specific aptamer on the surface-modified graphene at the channel area . Based oThus, the successful surface modification and immobilization of ADH-specific aptamers on the KOH-modified graphene surface were verified through ATR-FTIR characterization. The schematic illustration of the stages involved in the surface functionalization of graphene is illustrated in The electrical characteristics of the GFET device were monitored and measured as each individual surface functionalization step was carried out during the functionalization of the device a. Figure3-point probe measurements were carried out and changes in drain current in the range of 6 to 8 V were observed a. At thiThe value of This drastic decrement of orted by . The drain current, The increase of drain current in conjunction with the increase of ADH concentration clearly authenticates that the binding of positively charged ADH hormone with ADH-specific aptamer increases the number of positively charged biomarkers at the surface of the transducing channel. This, in turn, exerts an attractive force to attract negatively-charged charge carriers to accumulate in the channel area. Thus, the concentration of electrons increases in the transducing channel area (the area between the source and drain electrodes). This, in turn, increases the conductivity of graphene which is proved by the increase of drain current in the biosensor. This increase in ADH concentration leads to the increase of concentration of charge carriers in the transducing channel which causes a significant increase in c + 0.0009) with a correlation coefficient of 0.9613. Therefore, based on the slope of the calibration curve, the sensitivity of this biosensor to detect ADH spiked in PBS buffer is 5.00 × The graph of  plotted c. The liThe graph of the ratio of relative change in drain current versus logarithmic concentration of ADH spiked in PBS buffer is illustrated in The limit of detection (LOD) was considered with the lowest concentration of an analyte  against the background signal (S/N = 3:1), in other words, LOD = standard deviation of the baseline + 3σ. Based on 3σ calculation, the ADH biosensor exhibits a limit of detection as low as 3.55 ag/mL. LOD in the range of Therefore, LOD as low as 3.55 ag/mL shows an improvement in the detection limit compared to the research work that has been previously reported ,45,46. TThe specificity analysis on the ADH biosensor was carried out by dropping different biomolecules independently on the ADH-specific aptamer-immobilized transducing channel . The graGlucose oxidase (GOx) is an enzyme conventionally found in the human body, whereas urease is also an enzyme that could not be synthesised by mammalian cells. However, it can be produced by myriad bacteria found in the body, particularly in the intestine . The intFinally, human serum without ADH was pipetted on the functionalized transducing channel and the drain current was recorded. Based on When 1 pg/mL of ADH spiked with human serum was tested on the ADH-specific aptamer immobilized transducing channel, the drain current reading was recorded after a wait of 10 min. The drain current was then increased up to 557.89 µA when it indicated the successful binding of ADH-specific aptamer with ADH-spiked in human serum. This strongly suggested and proved that ADH biosensor is highly specific and selective. ADH spiked in PBS buffer and human serum demonstrated a large change in current and recorded a higher current compared to other tested biomolecules. This shows that specific interaction only takes place with ADH-spiked in PBS buffer and ADH-spiked in human serum which increases the charge carrier density at the transducing channel and eventually leads to a drastic change in current. Therefore, the ADH biosensor is highly selective and specific in interacting with ADH molecules which is promising for future applications in an artificial kidney.In conclusion, the ADH biosensor has a sensitivity of 50.00 µA·