In this study, the filled natural rubber (NR) was prepared with organic montmorillonite (OMMT) and carbon black (CB). The effects of the amount of OMMT on the properties of CB/NR composites were investigated by measuring the physical and mechanical properties, compression set and compression heat properties, processing properties and damping properties. The formulation was optimized depending on the different conditions of end applications and the damping properties of rubber were maximized without affecting the other properties of the rubber. The results showed that the rubber composite system filled with 2 phr (parts per hundreds of rubber) OMMT had better mechanical properties and excellent damping performance. With the development of rail transit, it has become a major problem to prepare high-performance  rubber shock absorbers to improve NVH  performance. Rubber can transform the mechanical energy to heat energy, and so can reduce or even eliminate the vibration, and has been widely used as a damping material ,2. In reTg), storage modulus, and the damping properties of composite materials. The results showed that the storage modulus increased with the increase of the dosage and the loss factor decreases gradually. Guo et al. [Wang  reportedo et al.  reportedo et al.  grafted o et al.  preparedOrganic montmorillonite (OMMT) has attracted extensive attention due to its good reinforcing effect, low price, abundant reserves, simple preparation and environmental protection. As a result, it is used as a new reinforcing filler in rubber. The influence of OMMT content on swelling behavior was investigated, showing that it can significantly improve the delayed expansion property of rubber . Chen etIt is rarely reported that OMMT was used to improve the vibration damping performance of rubber, while the layered structure of it is worth studying. At the same time, it can be found from the previous research that the damping performance of rubber is mainly affected by the structure of rubber and its filler. In this study, the processing performance, mechanical properties and damping properties of rubber composites were all improved only with little amount of OMMT. To characterize the damping performance of rubber material with OMMT (widely used as nanofillers in polymeric composites), the OMMT/CB/NR composites were obtained by mechanical mixing reaction intercalation method. In addition, studies on the influence of OMMT content and the composition of rubber components on its mechanical, dynamic mechanical properties and damping properties were performed. The characterization of the samples was done by rubber processing analysis (RPA), dynamic mechanical analysis (DMA), scanning electron microscope (SEM) and the mechanical analysis instruments.N-cyclohexyl thio-phthalimide , N-1,3-dimethylbutyl-N’-phenyl-p-phenylenediamine , Poly   and Zinc oxide (ZnO-80) were purchased from Ningbo Actmix Rubber Chemicals Co., Ltd, Ningbo, China The remaining ingredients were provided by Jinchangsheng Co., Guangzhou, Guangdong, China. Natural rubber  was used as basis material. Sulfur (99.5% purity) was purchased from Jinchangsheng Co., Guangzhou, Guangdong, China. Organic montmorillonite  was purchased from Zhejiang Fenghong clay chemical Co., Anji, Zhejiang, China. Cabon Black (N330) was purchased from Cabot Corporation, Tianjin, China. N-cyclohexyl thio-phthalimide, a kind of scorch retarder), 4020 , RD , a kind of antioxidant), mixed for 3 min. The following were carbon black N330 and OMMT, mixed for 3 min. After that, sulfur and ZnO (Zinc oxide) were added into the blended compound. Next, set the distance between the two rollers to 1mm, made the “triangle bag” in the process for 5 times. Lastly, the compound obtained was molded into sheets in a Type Plate Vulcanizer (XLB-400 × 400 × 2 50 T)  at 150 °C for 15 min.The formula of this study was listed in Mooney viscosity test was carried out on a CL-2000G Mooney Viscosity Meter at 100 °C  according to the standard of ASTM-D1646. Vulcanization performance was conducted using a MDR2000 Rotor Rheometer at 150 °C  according to the standard of ASTM-GB/T 16584-1996. Processing performance of each content were tested with three samples.1 according to HG/T 3849-2008, GB/T 529-2008 and HG/T 3321-2012. Hardness was measured by GT-GS-MB Shore A Hardness Tester  according to GB/T 531.2-2009. Compression set was tested according to GB/T 7759.1-2015 . Compression heat was tested by GT-RH-200  according to GB/T1687.3-2016.  at an impact speed of 14 m·s−1 according to GB/T 1681-2009. Five samples of each content were tested for mechanical properties.Tensile properties, tear strength and elasticity modulus were determined using AT-7000M Universal Electronic Tensile Machine  at a tensile speed of 500 mm·min⁻Dynamic rheological measurements of unvulcanized rubber compounds were carried out on a RPA8000 Rubber Processing Analyzer  by a shear mode at a temperature of 60 °C, a frequency of 1 Hz, and a strain amplitude in the range of 0.28–200%, according to ASTM D 6204. Three samples of each content were tested for data of RPA.The DMA was carried out using a DMA 242 Dynamic Mechanical Analyzer  in a double cantilever beam deformation mode within a temperature range of −80 to 80 °C with a heating rate of 3 °C/min and at a fixed frequency of 1 Hz. The experiment process was carried out in the atmosphere of nitrogen. Three samples of each content were tested for data of DMA.The micromorphology of OMMT in rubber matrix was investigated using a JEOL JSM-7500F scanning electron microscope (SEM) . Resolution: 1.0 nm at 15 kV; 1.4 nm at 1 kV. Acceleration voltage: 0.1–30 kV. One sample for each content was tested for SEM.L) increased gradually with an increase in the content of OMMT. It was determined by the viscosity of the rubber compound, which indicates the strength of physical interaction when the rubber is not cross-linked. Therefore, when the content of OMMT was increased, the physical interactions of the rubber gradually increased. The maximum torque (MH) increased appreciably with addition of 2 phr OMMT. However, the increment was not obvious with more amounts of OMMT addition. MH depends on the filler and the vulcanization system, and it reflected the superposition of physical interactions and chemical interactions after cross-linking. When OMMT was used in rubber, the MH-ML of compound increased, and the viscosity became larger thereby producing the agglomeration effect. Therefore, the larger content of OMMT, the stronger the agglomeration effect, and thus it damaged the mechanical properties of the material.Normally, the change in torque during vulcanization is proportional to the density of cross-linking including the physical and chemical cross-linking. The vulcanization properties of each content were tested. It can be seen from H and MH-ML as discussed in the above analysis.Hardness was measured at five different locations of each sample. It can be seen from In general, polymers are not used in their pure form, which means that the CB, silica and OMMT play an important role in the polymer reinforcement. The fillers in polymer strongly modifies the viscoelastic behavior, especially the dynamic mechanical properties of the composite. In dynamic mechanics measurements of shear modulus, the Payne effect is often referred to as the progressive breakdown of packing-packing interactions, where G’ is used commonly ,24,25.RPA8000 was used to conduct three-dimensional strain scanning on CB/NR compounds filled with different contents of OMMT, and the interaction between packing-packing and the interaction between packing-rubber were accurately characterized. Each content was tested with three samples and the experimental results were shown in In order to further study the dispersion of fillers in the CB/NR composites, we marked it in the RPA scans. The difference values of G’(1)-G’(3) at low strain could indicate the strength of the packing network, and the different values of G’(3)-G’(0) without adding filler at low strain could indicate the strength of the filler and the rubber . It can It can be seen from Tg of the OMMT/CB/NR composites slightly shifts toward the high temperature direction for the addition amount of 2, 4, and 6 phr OMMT. The slight increase of Tg is due to the fact that the rubber macromolecular segment is restricted by the sheet structure of OMMT and the exercise capacity is thus reduced. In the case of the 8 phr content, the agglomeration was produced and Tg was found decrease slightly, which is consistent with the analysis results of RPA. The peak value of Tanδ increased after OMMT was added, which was because the density of cross-linking within the vulcanized rubber increased. In the glass transition zone, the rubber was deformed by the external force and the flaky filler OMMT was oriented subsequently, which would generate friction with the rubber. This will transform the mechanical energy generated by the vibration into the heat energy loss, and then improve the damping of the material. As a result, the damping value of the material increased and the damping temperature range (Tanδ ≥ 0.3) was widened (As showed in  widened . FinallyThe CB/NR composite filled with 2 phr OMMT was found to have excellent mechanical properties and was up to the standard of the performance requirements of automobile damping vibration absorbing parts. Compared with the composite without OMMT, the crosslink density of the rubber was increased; the scorch time was extended; and the vulcanization rate was decreased. The OMMT is beneficial to improve the performance of the anti-vibration parts of the automobile and the processing safety. Meanwhile, the addition of OMMT could increase the dispersibility of CB and reduce the final temperature rise of compression heat generation. Both dynamic and static compression permanent deformations were quite small. After filling the OMMT, the system dynamic mechanical and damping properties were enhanced and the effective damping temperature range was broadened.


The release study of ACTH was tested using a modified USP apparatus 2 with Enhancer cells. The permeation study was conducted with vertical Franz cells. Rheograms of hydrogels were made with the use of a universal rotational rheometer. The dependence of the amount of released and permeated hormone on the ointment concentration was found. Based on the test of ACTH release from semi-solid formulations and evaluation of rheological parameters, it was found that glycerol ointment is the most favourable base for ACTH. The ACTH release and permeation process depends on both viscosity and ACTH concentration. The higher the hormone concentration, the higher the amount of released ACTH but it reduces the amount of ACTH penetrating through porcine skin.The transdermal route of administration of drug substances allows clinicians to obtain a therapeutic effect bypassing the gastrointestinal tract, where the active substance could be inactivated. The hormonal substance used in the study-corticotropin (ACTH)-shows systemic effects. Therefore, the study of the effect of the type of ointment base and drug concentration on the release rate and also permeation rate in in vivo simulated conditions may be a valuable source of information for clinical trials to effectively optimize corticotropin treatment. This goal was achieved by preparation ointment formulation selecting the appropriate ointment base and determining the effect of ACTH concentration on the release and permeation studies of the ACTH. Semi-solid preparations containing ACTH were prepared using Unguator CITO  Corticotropin (ACTH) is a hormone secreted by the anterior pituitary gland. It is a polypeptide consisting of 39 amino acid residues with a 4.5 kDa molecular weight ,2,3. It ® Gel, contains purified pig corticotropin and is administered by subcutaneous or intramuscular injection. This preparation is used in the treatment of, among others: West’s syndrome, bending attacks and mental retardation, usually diagnosed in the first year of life, an exacerbated form of multiple sclerosis, allergic conjunctivitis, ulcerative colitis, lupus erythematosus and vesicular epithelial separation  within 360 min. The release profiles was made based on obtained results.A modified USP Apparatus 2 equipped with 200 mL flat bottom vessels and mini paddles  was used for drug release studies. Excess amount of the ACTH ointment was loaded inside a special semisolid holder, Enhancer fluoropolymer cells (ERWEKA GmbH) with an exposed area of 3.8 cm2 values were calculated.The release results were fitted with different kinetics models such as zero order (% ACTH release vs. time), first order (log of % ACTH remaining vs. time), Higuchi’s model (% ACTH release vs. square root of time) and Korsmeyer-Peppas model (log of % ACTH release vs. log time). For each model R0-6h)-the area under the concentration-time-change curve was calculated. Area under the curve of concentration changes-time was counted as a numerical integral, using the parabol method. The size of the area under the concentration-time curve (AUC0-6h) was used to calculate the degree of relative availability  of ACTH 20 mg/g and ACTH 25 mg/g of ointment relative to the ACTH 15 mg/g reference, according to Equation (2):The availability of ACTH from the ointment (AUC2) between the ACTH concentration in the ointment and the total amount of released hormone after six hours and AUC were calculated.The obtained results were compared to the F-9 formulation, containing 15 mg/g of ACTH based on glycerol ointment. The existing interdependencies  with inner diameters 15 mm and permeation area of 1.77 cmon study . An accu2. Each of the formulations was applied to the skin surface.Porcine skin was used to study the permeation profile of ACTH due to the limited availability and difficulties associated with the use of ex vivo human skin. Nevertheless, porcine skin is a suitable alternative due to the similarities in thickness and permeability properties to human skin . The mod−1and 30 s−1, with shear stress measurement in relation to the shear rate (step by step) and a flow curve (flow test and hysteresis loops). Five repetitions were conducted for each experiment, and the mean values of the calculated parameters are presented (n = 5).Rheological tests were performed only for ointments releasing ACTH-for all hydrogels with corticotropin (F-7–F-9) and for hydrogel unloaded with ACTH. The analysis was performed using universal Reometry RM 200 Touch rotational rheometer by Lamy Rheology Instruments  for testing the rheological properties of samples. It was equipped with a CP1 Plus thermostat from Lamy Rheology Instruments with Rheomatic Lamy Instruments software. The tests were conducted in a system plate-plate geometry, using the MS CP 2445 Lamy Rheology Instruments measuring system with a diameter of 24 mm (α = 0.45°). Before measurement, the samples were placed in a CLW 53 STD incubator  at a temperature of 25.0 ± 0.5 °C. After 30 min, the sample was placed on the lower plate of the Peltier effect thermostatic system made of stainless steel, and the upper plate was lowered. The height of the gap between the plates was 50 μm. Excess sample was removed with a spatula. All rheological tests were carried out at 25.0 ± 0.5 °C. For the tested ointments, viscosity was measured at two shear rates, 15 s−1. The obtained results are presented in the form of shear rate dependence on shear stress as flow curves and also in the form of hysteresis loop for step-by-step test. The analysis was carried out in the tangential stress range 5.0–200.0 sp < 0.05 was calculated using Student’s t-test.Mean values with standard deviation were calculated and statistically analyzed using the Microsoft Excell package and Statistica  option: Industrial analysis, experimental design (DOE). The Statistica Pharmaceutical Kit: Statistica “Release Profiles” was used to analyze the results of ACTH release from the ointment bases. The Weibull method. Statistically significant significance at Analyzing the results of ACTH release test it can be stated that the degree of ACTH release depends on several factors: the base used, the concentration of ACTH, rheological parameters of ointment. Among the bases used, the most favourable ointment base for preparation of ointment from ACTH was hydrogel ointment (glycerol ointment), whereas the optimal concentration of corticotropin in the ointment was 20 mg/g. The higher the hormone concentration, the greater its amount was released into the acceptor fluid. The process of release at the latest began from ointment at concentration of 15 mg/g and as the concentration increased, more corticotropin was released at specific intervals in relation to ointments with the lowest concentration. The degree and rate of release of ACTH depend on the concentration of the active substance in the ointment and on the rheological properties of semisolid formulations. The addition of hormone causes an increase in viscosity in relation to the unloaded hydrogel itself. At the same time, a further increase in the concentration to 20 mg/g and 25 mg/g results in a increase in viscosity and release but increase in viscosity at 25 mg/g ACTH provide decrease in released amount of ACTH.Based on a study of corticotropin penetration through the natural membrane that is the skin of the pig’s ear, it was found that ACTH penetrates the skin. The inverse dependence of the amount permeated and the rate of hormone permeation on its concentration was obtained. The most advantageous formulation was the ointment with the lowest concentration , from which the corticotropin penetrated in the highest amounts and the fastest. With increased concentration and increased viscosity the amount of penetrating corticotropin decreased. These results do not confirm the relation obtained from the release test with the use of an artificial cellulose membrane. This fact proves different membrane properties and unpredictable character of processes taking place in the skin. Therefore, it is important to check the behavior of the active substance in simulated in vivo conditions. Based on the obtained results, it was found that there was a possibility of using ACTH in the form of ointments in clinical practice . Semi-solid dosage forms are a convenient form of skin application for potential patients, and unlike injections, a safe and non-invasive method of therapy.


The development of electrochemical biosensors is an important challenge in modern biomedicine since they allow detecting femto- and pico-molar concentrations of molecules. During this study, pillared graphene structures supported by vertically aligned carbon nanotubes (VACNT-graphene) are examined as the potential recognition element of DNA biosensors. Using mathematical modeling methods, the atomic supercells of different (VACNT-graphene) configurations and the energy profiles of its growth are found. Regarding the VACNT-graphene doped with DNA nitrogenous bases, calculated band structure and conductivity parameters are used. The obtained results show the presence of adenine, cytosine, thymine, and guanine on the surface of VACNT-graphene significantly changes its conductivity so the considered object could be the prospective element for DNA biosensing. The application of impedance technology has reduced the detection of complementary DNA sequence limits to 5 × 10−12 mol L−1 [−21 mol L−1 [−15 mol L−1 [DNA and microRNA play a key role in storing and transmitting genetic information. Currently, molecular analysis of DNA can diagnose more than 400 diseases . It is k mol L−1 . The dis mol L−1 , and for mol L−1 .−7 mol/L and a sensitivity of 53.89 μA mmol/L−1cm−2 for glucose detection with an excellent stability and reproducibility. An electrochemical chiral sensor via an integrated polysaccharides/3D nitrogen-doped graphene-CNT frame exhibited excellent reproducibility, stability, and selectivity [2-CNTs@GONR nanocomposite that exhibited satisfactory stability and accuracy for quercetin detection in actual samples [The high sensitivity of CNTs coupled with the larger detecting surface area of graphene makes the hybrid material on their base an ideal candidate for the role of a biosensor element. Zou et al., fabricated a novel glucose biosensor by loading a glassy carbon electrode with a 1-methyl imidazole-based ionic liquid-functionalized graphene/CNT composite . The bioectivity . Mousaabectivity . Three-dectivity . Zhao et samples .3 gas significantly increased the conductivity of CNTs [2O-graphene sheet nanostructures can be well used as FET to detect polycyclic aromatic hydrocarbons [Methods of mathematical modeling results serve as a guideline for the design and optimization of biosensors on the base of graphene and CNTs. Khodadadi et al., fabricated an epirubicin biosensor based on the interactions of DNA with polypyrrole and nitrogen-doped reduced graphene during the study of docking theoretical investigations confirming the binding of epirubicin and guanine sides in the ds-DNA structure . The ana of CNTs . This ef of CNTs . Studies of CNTs . To reve of CNTs . The simocarbons .−24 g), which is even better than the carbon nanotube-based nanosensors [Recently, such hybrid 3D materials as pillared graphene structures supported by vertically aligned CNTs (VACNT-graphene) have become increasingly popular. This material is successfully used for hydrogen storing and thermal transport and is used in nanoelectronic devices and supercapacitors ,37,38,39osensors . Here, mTo build atomic structures of VACNT-graphene, graphene nanoparticles with sizes 34.44 Å × 34.08 Å  and SWCNTs with chirality indexes , , , , ,  were used as the most often synthesized ,42,43. Ri − E0 was calculated, where E0 was the energy per atom of the initial GNM and Ei was the energy per atom of the current structure. Occurring at the next stage, the energy profile of the considered junction’s growth was constructed. To this aim, the author-developed technique called “virtual growing” was used, which was previously successfully tested for seamless junctions on the base of GNMs and SWCNTs of the armchair-type . RegardiThe SWCNT is the only tube with conductive properties among those considered, so the junction on its base was considered as the potential biosensor. Probably, the electronic structure of this particular compound will react more than others to the presence of other molecules near its structure. Since surface area and intertube bridging are key parameters for different properties of pillared graphene ,49,50, tSince the DNA molecule is too small for quantum chemical calculation, we performed a study of the interaction between the VACNT-graphene and nitrogenous bases: guanine, adenine, thymine, and cytosine often abbreviated by G, A, T and C, respectively. The search for the optimal location of nitrogenous bases on the surface of the VACNT-graphene supercell was performed using the SCC DFTB 2 method using the 16 × 16 × 16 Monkhorst–Pack sampling and 3ob-3-1 parameterization . As a ref = −0.4 eV, and for the composite decorated with adenine, thymine, or cytosine this value shifted to −0.24 eV. The exception was the addition of guanine—in this case the noticeable peak at −0.24 eV is observed. It also draws attention to the fact that the DOS of VACNT-graphene+A and VACNT-graphene+T are similar. This may be caused by the fact that adenine and thymine form a base pair in DNA [Band structure analysis showed that addition of adenine and guanine to the surface of the VACNT-graphene decreases its Fermi level from −4.44 to −4.87 eV, while the addition of just adenine lowers to −4.88 eV, and of just thymine further reduces to −4.89 eV. r in DNA .2/h). Similar to the case of seamless junctions between SWCNT and graphene of the armchair-type, the anisotropy of transmission is not observed [The electron transmission function T(E), electric resistance R, and electrical conductivity G were calculated at 300 K on the basis of the Keldysh nonequilibrium Green function method  and Landobserved . When thTo explain the presence of peaks on the graph of the integral characteristic T(E) , we calcAb initio calculations confirmed that band structures, as well as the conductive properties of this junction, were sensitive to the presence of the DNA nitrogenous bases near its surface. Absorption of guanine, adenine, cytosine, and thymine led to the appearance of the noticeable peak at the Fermi level where they significantly affected the composite’s conductivity. When the current flew along the zigzag directions, the presence of the DNA nitrogenous bases increased the conductivity of the VACNT-graphene by 5–10%, while, when the current flew along in the armchair direction, its conductivity dramatically decreased by 38–45%. The obtained results indicate that the VACNT-graphene composite may be a prospective candidate for the role of a recognition element in electrochemical biosensor of DNA. The future task is to provide studies of interaction between VACNT-graphene and large DNA molecules. Since the typical DNA molecules are large, the sizes of VACNT-graphene should be enlarged too. Our preliminary calculations, for example, showed adsorption of B-DNA dodecamer [The atomic supercells of VACNT-graphene with SWCNTs of the most often synthesized chirality indexes , , , ,  were obtained using the original molecular modeling methods. Our calculations showed that the growth of all considered structures was energetically favorable, which opens a prospect for its potential manufacturing. Containing semiconductive tubes, the VACNT-graphene structure is a prospective candidate for the role of a biosensor element. Its geometrical structure facilitates adsorption of different molecules on its surface. The first place for such absorption is located in the area of defects formed at the graphene and CNT junction during growth. Another place was found in the pores of CNTs. The presence of conductive CNT and superconductive graphene allows the assumption that conductive properties of the composite can be changed by adsorption of different molecules. odecamer  on the s


A suitable way to modify the electronic properties of graphene—while maintaining the exceptional properties associated with its two-dimensional (2D) nature—is its functionalisation. In particular, the incorporation of hydrogen isotopes in graphene is expected to modify its electronic properties leading to an energy gap opening, thereby rendering graphene promising for a widespread of applications. Hence, deuterium (D) adsorption on free-standing graphene was obtained by high-energy electron ionisation of D One of the most pursued key goals in graphene functionalisation is the achievement of a two-dimensional 2D) material with unvaried mechanical strength and stiffness but with different and tailored electronic and transport properties. In fact, while the extremely high mobility of graphene renders it appealing for applications, its semi-metallic nature represents a hindrance. Graphane is the forecasted semiconducting ,2,3 formD materiagraphane . Graphangraphane ,5,6, cougraphane ,8,9,10,  project ) has bee project . The sta project . Since TAll these potential outcomes of graphane share the need to incorporate significant hydrogen (or its isotopes) quantities in the carbon mesh. Several experiments have been carried out till now, mainly using hydrogen as the element to functionalise graphene, which also presents very similar chemical reactivity to that of deuterium. Graphene hydrogenation can be achieved by different methods, ranging from hot ,14,15 ancracking ,18; the cracking , to chemcracking ,20,21, tcracking ,23,24. TThe upper limit for hydrogen (deuterium) uploading can be due both to the presence of defects/edges (in 2D graphene flakes) or to the influence of the substrate . To overcome these drawbacks we propose to expose to deuterium high-quality, three-dimensional (3D), warped, free-standing graphene. Here, we study the effect of deuterium incorporation in nanoporous graphene samples: NPG is constituted by a compact, bi-continuous interconnected 3D arrangement of high-quality graphene veils, with 1000 m fermion ,26, comp fermion ,28. The  fermion  associat [3 bond . Thus, i [3 bond , by measIn this paper, after characterising and describing how to prepare a properly clean NPG sample, we describe its direct exposure to a deuterium ion beam into a deuterium atmosphere, with the ions generated by the irradiation of the gas with 1.8 MeV electrons; the Drgy ions . Furtherrgy ions .Nanoporous graphene was synthesised by using a nano-porous Ni-based chemical vapour deposition (CVD) method ,33,34,35Deuteration of NPG was achieved at the CIEMAT laboratory in Madrid . This inThis chamber was mounted in the beam line of a Van de Graaff electron accelerator. A 0.05 mm aluminium foil separated the gas chamber and the accelerator beam line in vacuum. In this way, 1.8 MeV electrons passed through the aluminium foil into the gas chamber. Once in the gas chamber, the 1.8 MeV electron beam was collimated in such a way that it passed between the two copper electrodes without touching the electrodes or the sample, just ionising the deuterium gas between the two electrodes without irradiating the sample. Once the deuterium gas was ionised by the 1.8 MeV electron beam, due to the action of an electric field perpendicular to the beam, free electrons generated by the ionising beam drifted to the grounded electrode and deuterium positive ions were directed to the negative (−500 V) electrode, where the NPG sample was placed. The ion density current reaching the graphene sample was measured during the experiment, being 1.4 mA/cmAfter deuteration, the D-NPG sample was eventually transferred in dry ambient conditions to the XPS chamber.The XPS and ultraviolet photoelectron spectroscopy (UPS) experiments were performed at Sapienza University in Rome, in the Nanostructures at Surfaces laboratory. The UHV chambers had a base pressure in the high 10tails in ,41,42,43The Raman experiments were carried out at the Optical Spectroscopy of Nanostructured Materials laboratory, at Sapienza University of Rome. As the excitation source, a Nd:YVO4 laser at 532 nm (DPSS series by Lasos) was employed. The measurements were acquired in a backscattering configuration, by focusing the laser and collecting the signal with an Olympus 50× objective (NA = 0.50). The laser power was kept below 100 W measured at the entrance of the objective to avoid sample damage, having checked that no changes were induced in the spectra with such a power, and that powers higher by about one order of magnitude were needed to induce possible damages. A 200-mm focal length monochromator (Isoplane 160) equipped with a 150 or 300 grooves/mm grating was employed to spectrally analyse the Raman signal, which was detected by a back-illuminated Si CCD Camera (model 100BRX by Princeton Instruments). Rejection for elastically scattered laser light has been achieved by a sharp high-pass Razor edge filter at 535 nm (Semrock).Scanning electron microscopy (SEM) images of NPG at the In order to overcome the limitations in hydrogen/deuterium incorporation, a cleaning protocol should be adopted, so to minimise defects and contamination of NPG samples. In fact, both the synthesis and the deuterium upload procedures, despite being carried out in controlled and the cleanest possible conditions, led to slight yet unavoidable oxygen contamination. A proper preparation procedure of the pristine NPG sample before exposure to deuterium can be achieved by annealing at 600 The NPG sample microscopically appears with an apparent non-homogeneous texture by optical microscopy a, while As we will discuss in the following, the Raman spectra acquired in our NPG samples show a very slight presence of defect/disorder related peaks, typical of a very high-quality and low-defect one-to-few layer continuous graphene sheet with a turbostratic stacking, thereby having only weak bonding between the sheets, as discussed in previous papers ,28.NPG was measured by XPS on pristine samples and after an annealing procedure in UHV, aiming at reducing possible residual contamination. The C 1s and the O 1s core levels as functions of annealing temperature and time are displayed in Ox bonds ,45,46, dResidual oxygen is the only intense contaminant present, with a concentration of ≥20 at.% estimated by taking into account the relative excitation cross sections of the O 1s and C 1s core levels , as showhisation . This clhisation . We remaRaman spectroscopy represents a non-invasive, all-optical technique particularly effective to probe the formation of new bonds with other atomic species, to achieve information on the number of layers, or to verify the presence of defects, doping, strain and lattice deformation in graphene ,51,52 aned domes ,58,59. Ied domes . In the To have a comprehensive characterisation of the deuterated sample, Raman measurements were performed both before and after the D-exposure. Raman spectroscopy has been by far among the most effective and well used techniques for estimating graphene lattice quality . The Ram K point . Further K point . However K point ,16,60,61In Both the G peak at 1578 cmh higher ,16,60,61The C 1s core level spectra of the D-NPG sample as-adsorbed and after subsequent temperature annealing steps, along with the data of the UHV-clean one for comparison, are plotted in The experimental data were fitted with components representing the different chemically shifted environments, after subtracting a Shirley background. In particular, we used the asymmetric Doniach–Sunjic (DS) lineshape for the main sper ≤ 0.1 ,63,64), The most prominent component of the C 1s spectra is associated with the speriments ,28. Theritations ,65,66,67urations ,45,46. Wurations ,69,70,71Deuterium uptake induces the following changes in the C 1s lineshape: (i) increase of the spcase see . We can position . Anothersorption . After t0 ∘C see . FurtherThe adsorption of H on graphene is known to turn graphene from a semimetal to a semiconductor ,2,21,72.In the UHV-clean NPG we observe the typical electronic spectral density of graphene, characterised by (i) the linear behaviour towards the Fermi level (Eσ−π band , evidencgraphene .The observed gap opening is further proof of the achievement of semiconducting graphane after chemisorption of D, in unprecedented, highly D-uploaded graphene with very high quality. We underline that the high percentage of spNanoporous graphene has been efficiently functionalised with deuterium atoms by Ddetector .


After the EFA; the varimax rotation method was employed for a factor through commonality analysis; reducing the 14 initial factors to 10. Only question 30 presented commonality lower than 0.5; and the other questions returned values higher than 0.5 in the commonality analysis. Regarding the reliability of the instrument; all of the questions presented reliability as the values varied between 0.953 and 0.956. Thus; the instrument demonstrated construct validity and reliabilityThe purpose of this study was to validate the construct and reliability of an instrument to assess the work environment as a single tool based on quality of life (QL), quality of work life (QWL), and organizational climate (OC). The methodology tested the construct validity through Exploratory Factor Analysis (EFA) and reliability through Cronbach’s alpha. The EFA returned a Kaiser–Meyer–Olkin (KMO) value of 0.917; which demonstrated that the data were adequate for the factor analysis; and a significant Bartlett’s test of sphericity ( A dynamic work environment marked by fast and continuous changes, both in social relations and work transformations, demands that managers and companies rethink their positions regarding the workplace, work methods, and the elements that compromise the realization of work activities by the worker adequately . The worUnderstanding the complex work environment is quite valuable for organizations, as it enables them to elucidate situations that generate health problems and consequently entail high absence rates and turnover, and low productivity at work. In this context, managers’ responsibility for the health and welfare of the workers has increased ,7. EnvirHealth-focused environmental assessment is conducted through research instruments or tools, investigating situations that might compromise workers’ health and consequently affect productivity at the workplace. Assessing this environment is a complex task, seeing that it comprises both micro-environmental  and macro-environmental  factors, which influence an organization’s progress .These factors in the work environment include quality of life (QL), quality of work life (QWL), and organizational climate (OC). Health condition is a fundamental aspect of QL and work capability , and a pSimilarly, QWL is seen as the degree in which an individual satisfies their personal and work needs through participation at the workplace . It is rFinally, OC is linked to motivation, loyalty, and the identity of each company . As collIt is noteworthy that the human being is complex and holistic, and therefore seeks in an individualized way to satisfy his needs. The theoretical concept of the three focus areas of this study, namely, QL, QWL, and OC, have the same strand and these focus areas are directly related to welfare, satisfaction in the workplace, and productivity, and when used in a unique way they become useful tools for managerial practice.In the current research scenario, work environment assessment tools or instruments focus on three areas: QL, QWL, and OC . They feIn this context, the applicability of these individual instruments becomes costly and challenging, as the investigation takes longer and the results cannot be compared and linked to other tools, thus returning unreliable responses. Therefore, the scientific gap for the development of this study reveals itself, with the realization that companies need practical and all-encompassing tools that provide more complete responses about the work environment.Taking that into account, this study’s general purpose was to validate the construct and reliability of an instrument capable of assessing the work environment as a single tool based on QL, QWL, and OC.This study is justified because, despite the existence of QL and QWL assessment instruments, their structures are based on classic theoretical models developed over two decades ago, specifically for the American labor culture . SimilarThe proposal of this new instrument model intends to fill this gap in the academic and organizational context. It will allow the researcher or manager to identify, with a single tool, the problems and/or situations in the work environment that might potentially compromise workers’ health and affect productivity, be it related to QL, QWL, or OC.The first element in the development of a research instrument is the construction of a conceptual structure  containing the questions and factors related to the theme . ObserviThe theoretical construct initially contained 23 factors and 83 questions. Afterward, a board of examiners composed of five experts  in the field assessed the instrument and suggested the removal of 9 factors and 23 questions, resulting in a final theoretical construct comprising 14 factors and 50 questions. The factors represented the areas encompassed by the instruments . Within them, factors represented by questions were created, aiming to assess the collaborator’s satisfaction.After establishing the instrument’s theoretical construct, its validation was started. The validity of an instrument is directly related to how it measures what it intends to measure . Thus, aThe validation of assessment instruments commonly employs the techniques of content validity, face validity, criterion validity, and construct validity . The valThe first step to validate the instrument was testing content validity, seeking to identify the clarity of language, practical pertinence, and theoretical relevance. This verification was conducted by evaluator-judges selected according to their field, following the procedures described by the authors : the insAfter content validation, a pilot test was applied to a group of 100 workers of the company Beta, the focus of this study, aiming to test the feasibility of the study’s design and the clarity of the instrument. In this moment all workers signed an Informed Consent, of ethics committee project CAAE 35553720.0.0000.5547. The test revealed that the workers understood the instrument, eliminating the need for changes.Subsequently, the construct validity was tested through exploratory and confirmatory factor analysis. To allow this analysis, the aforementioned company Beta, which employs 565 workers, was contacted and authorized the survey. For a reliability level of 95% and a margin of error of 5%, the necessary sample would be 229 workers. For its validation, the instrument was applied to 231 workers, belonging to the same group as the pilot study, at this moment, all workers signed the Free and Informed Consent Term, according to the Ethics Committee project CAAE 35553720.0.0000.5547. These company employees over 18 years old were selected, and those who would be excluded would be given incomplete or blank questionnaires; however, no questionnaire was given this way.r values are distributed as follows: r = 0–0.25, very low correlation; r = 0.26–0.49, low correlation; r = 0.5–0.69, moderate correlation; r = 0.7–0.89, high or strong correlation; r = 0.9–1.0, very high or very strong correlation [In order to assess construct validity, Pearson’s correlation was employed. Pearson’s correlation is commonly used to verify the intensity of the existing linear association between variables and it measures the linear association between quantitative variables . This corelation . PearsonThe next step was reliability analysis through test–retest of the instrument and analyzing its internal consistency. Reliability or trustworthiness seeks to identify how consistent are the scores obtained by the same subjects surveyed when they are re-examined, which occurs through the application of the same instrument on another occasion .The test–retest procedure was employed to verify the instrument’s reliability. The instrument was applied by the researcher in the same company Beta and the workers answered the questions (test). After seven days, it was reapplied to the same group (retest).The instrument’s reliability was verified through Cronbach’s alpha, calculating the existing correlation between each item of the test and the remaining items or the total . Cronbach’s alpha has particular characteristics that seek solutions in a population surveyed, not simply a measure by itself, and these alpha values change according to the group in which the measure is adapted .Having verified the instrument’s reliability, the next step would be the criterion validation, aiming to examine whether the measuring scale was adequate to what was expected in relation to other variables, which are selected through expressive criteria ,39. For Finally, a sensitivity analysis sought to ascertain whether the instrument was identifying its actual goal. The sensitivity analysis aimed to identify whether this study actually measured its intended objective among the population assessed and to detect significant changes in the construct studied .The instrument’s sensitivity was assessed through indicators of central tendency and dispersion  of both sexes, only men, and only women so as to compare the results. These data were obtained in the application of the instrument during the test stage, and they revealed that the instrument measured its intended target.The stages that required statistical assessment were performed through SPSS 22.0  for Windows.In order to validate this instrument, a survey was conducted with 231 collaborators of the aforementioned company, the focus of this study. The anthropometric data showed that the respondents were, on average, 32.3 years old (SD ± 10.91). The sample contained 139 male respondents and 92 female respondents. In terms of educational background, 9 had basic education, 70 had a high school degree, 42 were undergraduate students, 53 had a college degree, and 57 had post-graduate specializations. These 231 collaborators were distributed in the three hierarchical levels of the company . Within the questionnaires, information about the organization’s hierarchical levels was collected, but it did not include this as a demographic variable in order for this instrument to meet all hierarchical levels. Also pointed out that the hierarchical pattern was implicit in the educational pattern and that this varied from company to company.Thus, the anthropometric data revealed that the respondents were 32 years old on average, mostly male, had a high school degree, and were mostly single.χ² = 7465.349, Df = 1225, p ≤ 0.000), allowing the EFA to be conducted.The Kaiser–Meyer–Olkin (KMO) index, used to verify the suitability of the application of Exploratory Factor Analysis (EFA) for this study’s data set, returned a value of KMO = 0.917, which demonstrated that the data were suitable for factor analysis. Bartlett’s sphericity test was significant .The number of factors estimated was determined by assessing the scree plot, employing question retention through the Kaiser–Guttman criterion of the components with eigenvalues higher than 1 . The anaSeeking to test the unidimensionality of the instrument, the EFA was conducted for one factor through commonality, employing the principal component method. Only question 30 presented commonality lower than 0.5. The others presented values higher than 0.5, demonstrating the existence power of each question. According to , the higAfter the commonality analysis employing the principal component method, the component matrix was verified. Employing the principal component analysis as the extraction method and the varimax rotation method with Kaiser normalization, the instrument went from 14 factors initially to 10 factors after the statistical analysis.The 50 questions were distributed into 10 factors, each allocated in one or more domains. However, the extraction and rotation method revealed the highest score in the factors, determining in which one the question must remain. It was possible to observe that factor 1 was composed by questions 22–25, 28–30, 39, and 43; factor 2 by questions 26, 27, 31–33, 45–47, and 50; factor 3 by questions 4, 8–11, and 18; factor 4 by questions 34–37, 41, and 42; factor 5 by questions 1–3, 5, and 6; factor 6 by questions 15–17; factor 7 by questions 19–21; factor 8 by questions 38, 40, 44, 48, and 49; factor 9 by questions 12–14; and factor 10 was composed by question 7.The factors were named according to the area which they encompassed, as pointed by the instrument’s theoretical construct and displayed by Factor 1—Health comprised five questions . Factor 2—Emotional and Psychological comprised six questions . Factor 3—Spiritual comprised three questions (12–14). Factor 4—Sleep and Rest comprised only one question (7). Factor 5—Work and Life comprised three questions (15–17). Factor 6—Work Conditions comprised three questions (19–21). Factor 7—Leadership Management comprised nine questions . Factor 8—Remuneration and Functional Assistance also comprised nine questions . Finally, Factor 10—Personal Relations and Company Image comprised five questions .Seeking to analyze the construct validity of the instrument, the correlation matrix demonstrated the existing relationships between the pairs of variables.In the EFA application, Pearson’s correlation was conducted in the test–retest stage of the instrument, adopting a significance level of 0.01. The analysis of the correlation matrix revealed that all of the 50 questions presented a positive correlation.r values from 0 to 0.25 indicate a very low correlation, from 0.26 to 0.49 indicate a low correlation, from 0.5 to 0.69 indicate a moderate correlation, from 0.7 to 0.89 indicate a high or strong correlation, and from 0.90 to 1.0 indicate a very high or very strong correlation. The results showed that none of the questions presented very low correlations. Eight questions presented low correlations, thirty-eight presented moderate correlations, and four presented high or strong correlations, as displayed in According to , the r vThus, the 50 questions of the instrument were correlated. Even though eight questions presented a low correlation, they were significantly correlated.In order to determine the instrument’s internal consistency in terms of reliability, Cronbach’s alpha was calculated for the existing correlation between each item of the test and the remaining items or their total . The Cronbach’s alpha of the 50 questions is exhibited in Although determinant values higher than 0.7 are considered ideal , values The Cronbach’s alpha values presented in the instrument ranged 0.953–0.956. The only item that obtained 0.956 was question 5, while the others stayed within the interval mentioned.The results showed that all of the factors presented values higher than 0.7, which indicated the reliability of the proposed instrument.The full instrument is available in the χ² = 7465,349, Df = 1225, p ≤ 0.000). The EFA composed of the total of variables in the instrument revealed that nine questions should be removed from the initial fourteen. For that purpose, an EFA was conducted for one question through the commonality analysis, which presented only one question under the necessary parameter. However, this question remained in the instrument because it was the only one that addressed the theme of autonomy granted by the company to make decisions.The construct validation was conducted through Exploratory Factor Analysis, obtaining the values for KMO (0.917) and Bartlett’s test (After the commonality analysis, the component matrix was observed. The instrument, which at first featured fourteen factors, contained ten factors after the statistical analysis.Seeking to analyze the construct validity of the instrument, the correlation matrix was examined in the application of the instrument’s test–retest. All of the questions of the matrix presented positive correlations. Eight questions presented low correlation, thirty-eight presented moderate correlation, and four presented high or strong correlation.Regarding reliability, Cronbach’s alpha was calculated in order to determine the instrument’s internal consistency, pointing to values ranging from 0.953 and 0.956, which indicated that all of the questions presented reliability.Thus, it is possible to conclude that the proposed instrument is valid and applicable, seeing that it met all of the parameters necessary for its validation. It assesses elements of QL, QWL, and OC as a single tool that allows managers to comprehend the situation of the work environment.


The area of space immediately around the floral display is likely to have an increased level of humidity relative to the environment around it, due to both nectar evaporation and floral transpiration. This increased level of floral humidity could act as a close-distance cue for pollinators or influence thermoregulation, pollen viability and infection of flowers by fungal pathogens. However, with a few exceptions, not much is known about the patterns of floral humidity in flowering plants or the physiological traits that result in its generation. We conducted a survey of 42 radially symmetrical flower species (representing 21 widely spread families) under controlled conditions. Humidity was measured using a novel robot arm technique that allowed us to take measurements along transects across and above the floral surface. The intensity of floral humidity was found to vary between different flower species. Thirty of the species we surveyed presented levels of humidity exceeding a control comparable to background humidity levels, while twelve species did not. Patterns of floral humidity also differed across species. Nevertheless, floral humidity tended to be highest near the center of the flower, and decreased logarithmically with increasing distance above the flower, normally declining to background levels within 30 mm. It remains unclear how physiological traits influence the diversity of floral humidity discovered in this survey, but floral shape seems to also influence floral humidity. These results demonstrate that floral humidity may occur in a wide range of species and that there might be greater level of diversity and complexity in this floral trait than previously known. Some floral traits are difficult for humans to identify but nevertheless influence floral function, or are detectable to floral visitors . Floral Digitalis, Echium, and Helleborus flowers  was mounted in the center of the sampling zone. A scale floorplan for this room is given in All floral humidity measurements were taken within a 6.11 m × 3.67 m room within the Bristol University Life Sciences Building (51°45′N 2°60′W) that had temperature and airflow controls for maintaining a constant background temperature and humidity. A 3.72 m × 3.67 m section of this room, hereafter referred to as the ‘sampling zone,’ was separated off within a 2 m high wall of 10 mm thick clear polycarbonate. A six-axis articulated The background temperature was kept constant at 23.01 ± 1.38°C , as the amount of water vapor indicated by a relative humidity value varies depending on air temperature . For exaArduino UNO microcontroller . The Arduino transmitted the measurements via a USB port to a PC located outside the sampling zone (behind the polycarbonate screen). Two probes were used in measurements: a ‘background probe,’ which measured background humidity, and a ‘focal probe,’ which measured the humidity in the headspace of the object being sampled. The focal probe was mounted on the robot as detailed in All humidity measurements were taken using DHT-22 humidity probes  attached to an 3 horticultural tubes was fixed along one length of the table (A 75 cm × 90 cm × 74 cm (width × length × height) wooden table was placed in the sampling zone. A rack for 24 cmhe table  where hohe table  in order–1) and paused before starting to record measurements to allow any minute air disturbances to settle. Overall, the robotic arm was also unlikely to change the intensity and gradients of humidity produced by the sampled flowers.The two microcomputers registering the values from the focal and background probes were at a distance of 34.5 and 32.0 cm from their respective probes. They did not heat up significantly or possess any elements that might have generated air turbulence, whilst all elements relating to the control of the robot arm and the data-storing PC were located behind the polycarbonate screen . This me3 plastic horticulture tube. Horticulture tubes were filled to a point 2 cm from the tubes’ lid to provide the flowers unrestricted access to water. Cut flowers were then taken into the lab in these tubes, and measurements started quickly after arrival. If flowers were grown outdoors, care was taken to collect them in dry conditions, so that no standing water from condensation or rain could influence the measurements. Flowers were only used if they were fully open and did not show signs of age, disease or damage.To separate floral humidity from other sources of humidity on the plant, flowers were cut and placed in horticulture tubes prior to the start of the measurements. Furthermore, cutting allowed us to control the orientation of flowers during sampling . Flowersblu tack: Bostik, Paris, France) stuck to the tube lid  was 11:01 h, ranging from 9:10 h to 14:00 h. A full list of the times of the first measurements taken on each individual flower and control are given in All humidity measurements were carried out autonomously by the robot after moving the focal probe to predefined measurement positions in a predefined order, creating a transect-like sequence of humidity measurements. As a first step on a sampling day, the measurement positions of the focal probe had to be adjusted using manual controls, in order to account for different sizes and shapes of flowers. Thus, once flowers were ready for sampling, the focal probe was maneuvered to a ‘transect central point’ above each flower – the point above the center of the flower, 5 mm higher than the flower’s highest point. The transect central point for each flower and a single point less than 2.5 cm of the background probe were stored in the robot’s memory. Once a point is stored in the robot’s memory, the robot can return the focal probe to this position, and positions relative to them, in a predefined sequence.x axis movements are horizontal, moving toward and away from the robot ; z axis movements are up and down . The axes of the robot relative to the room and the table in the sampling zone are indicated in The sequence order in which individual flowers were sampled on each day was selected via a randomization algorithm within the arm’s software. The robot began each sampling sequence with the focal probe to a ‘safe’ position 50 cm above the table . In the x axis, and then along the z axis, relative to the flower  move to the safe position, (2) move to the flower’s or control’s position, start transecting and sampling humidity along the e flower , (3) movx axis from that flower’s transect central point . Measurements were taken in 5 mm intervals along this transect . Humidity measurements occurred regardless of whether the probe was above the flower surface at a sampling point of not (most flowers sampled are less then 60 mm wide). This meant that, even though flowers differed in size, all humidity transects were still the same size, aiding comparisons of floral humidity between different flowers and species. Following completion of measurements of the x axis transect, the robot moved the focal probe to a position that was offset by 5 mm in the z axis from that flower’s transect central point . Again, measurements were taken in 5 mm intervals along this transect . This sequence of movements within the transects, as well as the direction of movements and the orientation of x and z transects relative to the flower and robot, was the same in all transects (as demonstrated in –1). A third (y axis) transect was deemed unnecessary because all flowers and inflorescences that we sampled showed high radial symmetry.In order to start taking measurements whilst transecting the headspace of a flower or probe, the arm moved from the safe position toward the flower or control to a point that was offset by –30 mm in the al point . This poal point . This povia the safe position when moving to and from a probe-control measurement was to avoid disturbances of the headspaces in the flower array.Following completion of both transects on a flower the robot would return to the safe position 50 cm above the table, then move the focal probe to the point near to the background probe (approximately at a distance 2.5 cm from it). Here, it would carry out a probe-control measurement for 230 s, as described below. Following completion of a probe-control measurement, the robot would move the focal probe back to the ‘safe’ position starting the sampling of the next flower or control in the predefined sequence. The movement Only one set of flower arrays was collected and measured per day. The initial randomized order chosen by the robot was maintained across all four replications of the measurement sequence on a sampling day. This meant that each flower was resampled four times within time intervals of 231–308 min. Following completion of the probe control measurement on the final flower in the sequence on the last replicate sample, the robot would return to the ‘safe’ position and power down. All raw data collected are available in At each sampling point along transects , to predict one probe’s measurements from the other when in this position. This was later used to adjust the ‘uncorrected focal relative humidity’ (funcorrected) values for other points usingProbes can vary slightly in their estimations of the same levels of humidity (±5% according to the manufacturer’s specifications). Thus, the probe-control measurements were necessary to calculate how much the probes differed in estimates and reduce this source of inaccuracy. Upon moving the focal probe next to the background probe, the focal probe waited for 30 s before measuring humidity for 200 s, and the background probe made simultaneous measurements. Assuming that both probes are measuring a point of the same humidity during the probe-control measurements we could later compute linear regression parameters, using the MATLAB ‘W and M are, respectively, the slope and intercept parameters obtained from regressing the focal probe measurements against the background probes measurements for the time period of the probe-control measurements. This focal probe correction (Eq. 1) was calculated and applied across each replicate sample of the flowers sampled on the same day . This was done to account for any possibility that the difference between probes may change over time.where To characterize between-species differences in flower size, a flower’s horizontal span was measured on fresh unsampled flowers against a rigid tape measure, or where not possible, estimated based on miscellaneous literature records (cited in the “Results” section). Fresh flowers or literature records, as opposed to measurements from the individual flowers sampled under the robot, were used to measure flower size as such measurement might disturb floral headspace and floral humidity production. Flower or inflorescence structure, where appropriate to each species, was classified based on species descriptions from n = 14) of the species surveyed. Petals were removed from fresh unsampled flowers, and a mold was made of the upper petal surface using dental wax . Again, fresh flowers were used to survey for stomatal presence, as opposed to those sampled under the robot, as such sampling may damage or disturb the floral headspace and floral humidity production. A cast was made from this mold using clear nail polish, and was mounted on a microscope slide using tape . Mounted casts were surveyed from petal base to tip using a light microscope at ×100 magnification, and the presence of stomata was recorded. A species’ presence of stomata was expressed as the percentage of petals surveyed that showed the presence of any stomata.The presence of petal stomata was surveyed in a subset (R 3.4.1 (fcorrected) and the simultaneous background measurements (fbackground) taken throughout the transects can be expressed as a relative humidity whereAll analyses were conducted in R 3.4.1 . The difRH > 0 an increase in relative humidity is detected by the focal probe.Where ΔRH, Eq. 2) was calculated within each measurement period . These averages at each measurement period, were then used for analyses of humidity structure. For each flower species (and control), a series of linear models describing different humidity structures were fitted to this x and z axis data, as described below. How well these different models described humidity structure was compared using AIC to identify the best-fitting model of x and z axis humidity structure for each species (and control). These best-fitting models were then used to estimate humidity intensity for each species (see below). The most complex model (the ‘full model’) fitted to x axis transect data of each species had a quadratic structure, the most complex model fitted to the z axis data had a logarithmic structure. These full models also allowed humidity to vary between repeated transects. All other models represented simplified versions of the full models. These quadratic and logarithmic models were chosen over more complicated curve fitting methods and functions as, in addition to their relative simplicity, these functions easily condense to other structures as models are simplified . This allows the identity of best-fitting models to indicate differences in floral humidity structure, and changes in humidity over repeat transects. Throughout all floral humidity models, flower identity was included as a random factor influencing the intercept, or amount of humidity produced.The mean difference between the focal and background probe  within rptR 0.9.21  measurements taken during the first 20 s and the last 20 s at each measurement period, denoted The turbulence generated by the movement of the probe may cause humidity to not settle by the end of the 30-s settling time, which could cause a change in humidity measurements at the focal probe between the start of a measurement period (where remaining turbulence would be greatest), and the end (where it would be most stable). It is possible this difference may differ dependent upon the humidity at a sampling point: if little water is present in the air, disruption of that area may affect humidity estimates less than more humid air. To assess the effect of remaining turbulence, the change across a measurement period should therefore be assessed while accounting for how humid the disturbed area is. Furthermore, the directionality (positive or negative) of this change in humidity may differ depending on how the air mixes as a result of turbulence and the humidity of air being mixed in. If turbulence brings in more humid air, humidity may be raised, while the reverse might be expected if less humid air is brought in. To avoid turbulence effects being obscured by conflicting (positive or negative) changes in humidity, the non-directional change in humidity across a measurement period should be considered, rather than just the change in humidity. The presence and extent of remaining turbulence after the settling time was assessed by calculating the means of the focal humidity (c. 100 measurements), x axis transect of a given species or control isThe full model fitted to the X refers to the x axis offset of the sampling point (from –30 mm to +30 mm in steps of 5 mm) relative to the transect central point (where X = 0), t varies from 1 to 4, as the sampling sequence was repeated four times for each flower or control: 1 being the first run of a sampling sequence; 2, 3, and 4 being the second, third, and fourth repetitions, respectively. ΔRHxnt is the mean ΔRH for the measurement period at sampling point X, on transect repeat sample t, on flower n. Ax and Bx are parameters that describe the positioning and slope of the x axis humidity profile in the initial transect. Parameter Ix is the model intercept in the initial transect. Ix is modified by random factor vxn, which represents the change in model intercept, and consequently the intensity of humidity generated, by individual flower (or control tube) n on the x axis transect. Further random factors accounting for individual variation in the shape of humidity structure were not included, in order to simplify models. As flowers may show changes in humidity produced over time across replicate transects, Ax, Bx, and Ix are also modified by parameters ax, bx, and ix, which represent the change in the offset, slope and intercept, respectively  between first and latter replicate transects. The values of ax, bx and ix within each replicate transect are modified as follows:where gtx, ctx, and rtx are the values of ax, bx, and ix during repeat sample transect t, whenwhere gtx, ctx, and rtx are the changes applied to Ax, Bx, and Ix in transect t. Here parameters Ax, Bx, Ix, vxn and all iterations of gtx, ctx, and rtx are parameters to be estimated. The x axis model described by Eq. (4) assumes a degree of symmetry in humidity structure, this is due to the high level of radial symmetry shown by all species sampled. Although some degree of asymmetry, off-center positioning, is included in the model (primarily through the effects of parameters Ax and ax).Consequently z axis transect isThe full model applied to the Z refers to the sampling point’s z axis offset relative to the transect central point  function in the same manner described for respective parameters in the x axis model. Here, parameters Iz, Bz, vzn and all iterations of ctz and rtz are parameters to be estimated.Here, parameter al point . All othx and z transects described in Eqs (4) and (11), as well as simpler versions of these models, were fitted to the x and z transect data of each species and each control group. Simpler models were achieved by removing certain parameters from the full models, done by forcing these parameters to have values of zero. Eleven x axis models and five z axis models were compared for each species. These models are summarized in x and z axis models, respectively. These models were then compared using AIC to select the best models for the x and z transects of each flower species following the guidelines within The full models for the x transect as predicted by the best-fitting humidity model, was calculated for each flower species. This x axis offset (parameter X) value that the best-fitting model predicted to give the largest difference in humidity on transect t, for each species. x axis model of each species. If the best-fitting x axis model was m0 or m4 . In species where the x axis showed a quadratic relationship in the best-fitting model, X value of the vertex of the quadratic curve described by Eq. (4) . Due to this a formula for x axis model can be derived, as detailed in To evaluate the intensity of humidity generated by each flower species and by controls r m4 see , a valuex axis model of each species.xn was set to zero:Once RHx, the In instances where replicate transects had an influence on ΔA phylogenetically controlled analysis was carried out to assess the extent to which floral humidity produced by species . This means that within each measurement period the majority of measurements are about the mean value. This confirms the robot’s consistency of measurements and validates our decision to use mean values of each measurement period for our analyses of humidity structure, as measurements within each period are largely similar.The robot arm showed high repeatability over each set of approximately 100 measurements made in the same measurement period  for each control are given in To detect potential sources of extraneous humidity, we conducted measurements of control samples, such as empty or water filled tubes without flowers placed inside the array . The besblu tack putty gives off a small amount of water as it dries out.When the horticulture tubes had no water in them we saw little change in humidity between focal and background probes, hence the low controls . This suThe addition of water to the controls increased humidity, as seen by the higher controls . This sh control . Humidit control . This isTaken together, it seems that at most humidity extraneous to the flowers from the horticulture tube will be similar to that of the TWL control . Frequently, there was increased variation in humidity about the center of individual flowers, with a positive skew, relative to the rest of the transect  had non-quadratic best-fitting x axis models, as shown in Bx in some way. The x axis humidity structures of six species are given in X = 0) for 22 species, Typically, the humidity structure detected in the al point .z axis were less consistent in structure. The z axis structures of six species are given in z axis models showed a logarithmic relationship with humidity declining with increased distance from the flower (increased z axis offset). In most instances humidity reaching background levels by the end of the 30 mm transect. Although 17 species favored a flat z axis model where humidity remained level across the transect (Humidity profiles in the transect  as seen r), but in 13 species  showed some changes in structure of floral humidity with replicate transects (interacting effects determined by g and c). In most species the humidity decreased in the later replicate transects  explained the data better than the model with BM correlation . The model parameter effects are summarized in p = 0.092).The fitted full phylogenetically controlled model with OU correlation . We note here that we deliberately excluded leaves from our survey, although these were considered with Calystegia silvatica). Varying levels of floral humidity between species may mean that humidity could provide sensory information that pollinators could not only use to determine the presence and location of rewards on a flower, but also potentially facilitate the discrimination between different flower types. The differing floral humidity may reflect different humidity demands on pollen viability, such as the presence of desiccation resistant pollen that may mitigate the need to elevate floral humidity  produced humidity of an intensity that exceeded these  at once. Such procedures would sample the floral headspace more completely and create more three-dimensional measurements of floral humidity structure. Our models assumed a degree symmetry in x axis humidity structure as all species sampled showed a high degree of radial symmetry. For the same reason we did not carry out a y axis transect. Our survey suggests radially symmetrical flowers generally show symmetry in their floral humidity, given x axis humidity structure is quadratic in most species (y axis). Similarly, more complex sampling procedures could be applied to investigate the occurrence of floral humidity in flower species that do not show radial symmetry. It would be expected that such differences in floral shape related to floral symmetry would lead to differences in floral humidity structure compared to radially symmetrical flowers, particularly the symmetry of floral humidity.The transects used in this survey only sampled partial cross-sections of the whole flower headspace  as oppos species . HoweverO. caespitosa were not too dissimilar from those reported by x axis humidity signal of approximately 4% . This would allow floral transpiration and nectar evaporation to occur in a manner more similar to how it would in an open environment, perhaps allowing a more natural humidity equilibrium to be reached. Furthermore, the arm was also capable of more complex motion, allowing us to conduct continuous probe control measurements throughout the experiment, reducing variation as a result of differences between probes.The measurements we made of espitosa , resultiespitosa . It coulespitosa . AlternaOur survey of floral humidity across different plant species has revealed that floral humidity can be detected in many species. Our transects showed that floral humidity intensity and humidity structure can vary between species, although the reasons behind this variation remain unclear. In this way, our transects reveal floral humidity to show a greater level of diversity and complexity than previously known. This wider occurrence of floral humidity could enhance our knowledge of plant-pollinator signaling, as floral humidity is a known cue for at least one pollinating species . SimilarHyles lineata . NH contributed to study conceptualization, funding acquisition, and writing (review and editing). HK carried out robot programming and initial operation. HW contributed to study conceptualization, funding acquisition, and writing (review and editing). SR contributed to study conceptualization, funding acquisition, carried out phylogenetic analyses, and writing .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Looking for goal-relevant objects in our various environments is one of the most ubiquitous tasks the human visual system has to accomplish Wolfe, . Visual  N = 85 observers]. We have recently referred to this as the “down-side” of spatial context learning  extract and encode repeatedly encountered distractor-target relations in spatial long-term memory, the latter likely involving the medial temporal lobes (MTL). Once established, these memories are retrieved automatically by the search display presented on a given trial, with the activated contextual (distractor-to-target) associations providing pointers to the target location (and thus enhancing its activity) within the search-guiding attentional-priority map. Once consolidated, these representations come to generate memory-related costs in situations requiring contextual adaptation, that is, when the target is consistently repositioned to some other, “new” location within an otherwise unchanged distractor layout: in this case, the distractor context points to the “old” target location, thus cueing attention to the “wrong” location. Given that these memory-based (mis-) guidance signals are triggered automatically, it takes extensive practice to overcome them  signal between the current display input and the expected, that is, initially learned, target position. This mismatch signal would eventually also promote new learning, that is, the adaptation of contextual cueing to the changed distractor-target relations. Recent evidence suggests that the brain indeed works along these lines. For instance, in a recent study  or the rate of change between these displays  “belief” about the general level of regularity prevailing in the current sensory environment, with potential implications for overcoming associative blocking and enabling contextual re-learning.One promising computational mechanism that potentially mediates contextual cueing is spike-timing dependent plasticity  adaptability of contextual cueing. The relevant findings suggest that the relational memory underlying contextual cueing should in principle be flexible and thus be able to incorporate target position changes into previously acquired context memories. For instance, a bulk of neuroscientific data suggest that contextual cueing is supported by the medial temporal lobe (MTL), and especially the hippocampus (HC; e.g., Chun and Phelps, Irrespective of the type of – HC and/or PHC – memory system supporting contextual cueing, a model of the memory-guided search must specify how and when context-based memories interact with basic visual-search processes. This question was addressed in a series of recent electrophysiological and patient studies showing that long-term memory for scenes aids visual processing very rapidly, with the earliest electrophysiological markers of guidance becoming evident only 80 ms post display onset at occipital electrodes contralateral to the hemifield of the target (Zinchenko et al., The phenomenon of contextual cueing illustrates that the human visual system constantly generates predictions from repeatedly encountered events and scenes in our environment that help us to rapidly detect and respond to critical target objects in familiar contexts (Chun and Jiang, We argue that, considered together, these perspectives can provide new impetus for research on the task factors that regulate the interplay of the two types of representation and the effects of more HC-dependent or more PHC-dependent representations on initial contextual learning and subsequent contextual adaptation (for first demonstrations, see, e.g.,: Lleras and Von Mühlenen, The studies involving human participants were reviewed and approved by the Ethics Committee of the LMU Department Psychology. Participants provided their written informed consent to participate in this study.TG, HM, and AZ conceived and designed the perspective article. WS and MC contributed to discussion. TG, HM, and MC wrote the paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this work, we report a novel method of label-free detection of small molecules based on direct observation of interferometric signal change in graphene-modified glasses. The interferometric sensor chips are fabricated via a conventional wet transfer method of CVD-grown graphene onto the glass coverslips, lowering the device cost and allowing for upscaling the sensor fabrication. For the first time, we report the use of graphene functionalized by the aptamer as the bioreceptor, in conjunction with Spectral-Phase Interferometry (SPI) for detection of ochratoxin A (OTA). In a direct assay with an OTA-specific aptamer, we demonstrated a quick and significant change of the optical signal in response to the maximum tolerable level of OTA concentration. The sensor regeneration is possible in urea solution. The developed platform enables a direct method of kinetic analysis of small molecules using a low-cost optical chip with a graphene-aptamer sensing layer. Development of label-free, highly sensitive, direct, and real-time-enabled optical biosensors for small molecules (less than 1 kDa in size) is a challenging task of high importance in many areas of health analysis, environmental and food control, agriculture, etc. .In the last decade, the graphene-coated surface plasmon resonance (SPR) interfaces have been actively studied for application in biosensing ,4. Graphwww.realsense.rs), aiming to develop sensors for optimization of a cultivated meat bioprocess, it is important to consider sensing options for mycotoxins as well, including OTA.Ochratoxin A (OTA) is one of the most frequently occurring mycotoxins found in a number of different food products, including meat, meat products, and edible offal . In the The cultivated meat bioprocess starts with the seed train of primary cells, obtained by a biopsy from the animal. Since the animal may have been already fed with mycotoxin-contaminated feed , it is iDetecting the changes in optical properties such as absorption, reflection, and an interferometric pattern in the presence of analytes is one of the prospective methods for mycotoxin detection. Recently, graphene-based refractive index optical sensors were demonstrated for detection of different analytes ,15. The p- polarizations of a single beam [However, interferometric methods are less sensitive to graphene integration and can provide easy functionalization of the sensing area , yieldingle beam . Biorecegle beam . Aptamergle beam . Howevergle beam ,21. It igle beam ,23.Most commonly, interferometric sensors use the competitive immunoassay format or the binding inhibition assay where the target is attached to the surface ,24. VariIn this work, we report, for the first time, the label-free optical detection of ochratoxin A (OTA) in a direct assay using graphene functionalized with the OTA-specific aptamer and implementing the method of spectral phase interference (SPI). The SPI method is based on measuring the phase shift in an interference pattern produced by the reflected light from different surfaces within the sensor chip . This me2 size. The graphene from the backside of the copper foil was etched in solution made of deionized (DI) water, hydrochloric acid (30%), and hydrogen peroxide solution . To remove the copper foil, the sample was etched in aqueous solution of iron (III) chloride for about two hours. Prior to graphene transfer, the surface of the glass cover slips was washed in boiled dimethylformamide (DMF) and activated in oxygen plasma for 5 min. PMMA/graphene stack was rinsed in deionized (DI) water, and transferred onto the glass cover slip. A chip was left under ambient conditions overnight to dry out. To reflow the PMMA and improve the graphene-to-substrate adhesion, we annealed the chip at 150 °C for 5 min [Single-layer graphene chemical vapor deposited (CVD) on 25-µm thick copper foil was provided by Graphenea (Spain). A poly (methyl methacrylate)  was spin-coated with a step-wise increase of the speed from 1500 to 2500 rpm for 1 min on top of the graphene/copper stack and used as a support layer during the transfer. The stack was baked on a hot plate at 150 °C for 5 min. We cut the copper/graphene/PMMA stack to pieces of about 4 × 5 mmor 5 min . Afterwaor 5 min .n-hydroxysuccinimide ester (PBASE) in dimethylformamide (DMF) was spread over the cover glass for 6 h in order to activate the graphene surface. Afterward, the glass was washed thoroughly with DMF, isopropanol (IPA), and DI water for 3 min each. At the last step, the glass was gently blown with the air gun. The OTA aptamer with sequence of GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA [via an n-hydroxysuccinimide cross-linking reaction [Aptamer immobilization on graphene was performed in a way previously described for graphene-based transistor . 100 µL  CGG ACA ,30 with reaction  was carr2 and laser power of 0.5 mW. A Solver Pro atomic force microscope (AFM)  was used to study the morphology of the graphene transferred onto the cover slip. The silicon semi-contact cantilevers NSG03  with resonance frequency of 105 kHz were used in order to avoid any damage to the graphene during characterization.Raman spectra were recorded on the Centaur HR microRaman spectrometer  with a 100× objective at a 532-nm wavelength  with a beam spot of ≈1 μmTo investigate the effect of the small molecules’ conjugations with the modified graphene, we dissolved the OTA solution  in PBS in different concentrations. We used a compact single-channel label-free biosensor without thermal stabilization based on the spectral-phase interferometry, which was powered and controlled via a notebook USB port   is relaWe employed the possibility of the measuring system to follow the kinetics of the binding process to control the aptamers’ immobilization on graphene functionalized by the π-π stacked PBASE. The glass/graphene stack was exposed overnight to a 300-nM OTA aptamer solution in PBS a. The imIn this research, we used the OTA mycotoxin (403.8 Da ) as an eτ was calculated from the time course of the sensogram for the 10-nM concentration of OTA (τ is depending on toxin concentration and rates of association and dissociation of the aptamer and OTA. The results of binding kinetics fitting according to Formula (1) give an approximation of τ ≈ 6 min for 10 nM, which is about 40% lower than previously recorded data for the OTA aptamer by methods of total internal reflection ellipsometry (TIRE) [The response time n of OTA b based oy (TIRE) . In TablBetween measurements, the sensor was regenerated by washing in urea solution. We observed a nonlinear response to OTA concentration with the limit of detection of OTA above 1 nM. The thickness changes occurring upon OTA binding to the aptamer are presumed as the main mechanism of the sensor response. The shift in Fermi level under adsorption or reconfiguration of molecules (aptamer) can be interfered by the thickness change in absorbing molecules as the aptamer transforms from the linear stack to the G-quadruplex  with an However, the response of graphene to small molecules can be even more complex. When the aptamer molecule is attached to the graphene, it will increase electrostatic doping by the negatively charged aromatic bases of the nucleic acid in the aptamer molecule. Furthermore, the conformational change of the aptamer after bonding to the surface leads to decrease in electrostatic doping and, hence, change in the conductivity of graphene . These cIn conclusion, we have developed a proof-of-concept for the robust and scalable methodology based on the interferometric optical sensors for direct, label-free detection of the small molecules, such as mycotoxins by aptamer on a glass surface. We prepared the cover glass/graphene stack as a sensor chip with the OTA-aptamer covalently bonded onto the graphene surface. Implementing a compact interferometric biosensor powered and controlled via a notebook USB port, we demonstrated the sensitivity on the level of 10 nM of OTA with an assay time of 6 min. This technology is very flexible and can provide the basis for a direct assay of various small molecules’ detection based on aptamer-functionalized graphene, potentially in a multiplex format. Given that the interaction of aptamers and small molecules can be registered via changes of the graphene optical properties, it opens the way for utilization of the assay in drug discovery, food, and medical analyses. The described graphene-based measurement system is a potential new tool for cultivated meat contamination assessment as well.