The Effects of Three-Dimensional Ligand Immobilization on Kinetic Measurements in Biosensors

The field of biosensing is in constant evolution, propelled by the need for sensitive, reliable platforms that provide consistent results, especially in the drug development industry, where small molecule characterization is of uttermost relevance. Kinetic characterization of small biochemicals is particularly challenging, and has required sensor developers to find solutions to compensate for the lack of sensitivity of their instruments. In this regard, surface chemistry plays a crucial role. The ligands need to be efficiently immobilized on the sensor surface, and probe distribution, maintenance of their native structure and efficient diffusion of the analyte to the surface need to be optimized. In order to enhance the signal generated by low molecular weight targets, surface plasmon resonance sensors utilize a high density of probes on the surface by employing a thick dextran matrix, resulting in a three-dimensional, multilayer distribution of molecules. Despite increasing the binding signal, this method can generate artifacts, due to the diffusion dependence of surface binding, affecting the accuracy of measured affinity constants. On the other hand, when working with planar surface chemistries, an incredibly high sensitivity is required for low molecular weight analytes, and furthermore the standard method for immobilizing single layers of molecules based on self-assembled monolayers (SAM) of epoxysilane has been demonstrated to promote protein denaturation, thus being far from ideal. Here, we will give a concise overview of the impact of tridimensional immobilization of ligands on label-free biosensors, mostly focusing on the effect of diffusion on binding affinity constants measurements. We will comment on how multilayering of probes is certainly useful in terms of increasing the sensitivity of the sensor, but can cause steric hindrance, mass transport and other diffusion effects. On the other hand, probe monolayers on epoxysilane chemistries do not undergo diffusion effect but rather other artifacts can occur due to probe distortion. Finally, a combination of tridimensional polymeric chemistry and probe monolayer is presented and reviewed, showing advantages and disadvantages over the other two approaches.

Kinetic frustration by limited bond availability controls the LAT protein condensation phase transition on membranes

LAT is a membrane-linked scaffold protein that undergoes a phase transition to form a two-dimensional protein condensate on the membrane during T cell activation. Governed by tyrosine phosphorylation, LAT recruits various proteins that ultimately enable condensation through a percolation network of discrete and selective protein-protein interactions. Here we describe detailed kinetic measurements of the phase transition, along with coarse-grained model simulations, that reveal LAT condensation is kinetically frustrated by the availability of bonds to form the network. Unlike typical miscibility transitions in which compact domains may coexist at equilibrium, the LAT condensates are dynamically arrested in extended states, kinetically trapped out of equilibrium. Modeling identifies the structural basis for this kinetic arrest as the formation of spindle arrangements, favored by limited multivalent binding interactions along the flexible, intrinsically disordered LAT protein. These results reveal how local factors controlling the kinetics of LAT condensation enable formation of different, stable condensates, which may ultimately coexist within the cell.

Linear and non-linear regression analysis for the biosorption kinetics of methylene blue

The nonviable biomass of Rhizopus sp. R-18, Penicillium candidum and Penicillium chrysogenum was studied for biosorption of methylene blue (MB). The sorption of MB was studied be performing batch kinetic experiments. Kinetic measurements showed that sorption of MB reached equilibrium in 4 hours. The batch sorption models, based on a pseudo-first, pseudo-second and pseudo-nth order were applied to predict the rate constant of sorption and the equilibrium capacity. The linear and nonlinear least-square methods were used to obtain the kinetic parameters. The best-fit model was identified using statistic analysis. The results showed that both linear and nonlinear form of pseudo- second order expression could be used to fit the experimental data but nonlinear method may be a better way to obtain the desired parameters. As well the pseudo n-th order kinetic model was successfully applied to the kinetic data. The order (n) of adsorption reaction was found for all employed biosorbents: for Rhizopus sp. R-18 it had value 3.1, P. candidum 3.0 and P. chrysogenum 3.8.

The Adsorption of Phosphate on Mica Surfaces

<p>The surface chemistry of the 001 face of cleaved mica sheets was studied with a view to understanding some of the fundamental processes underlying the phenomenon of fixation of phosphate by soils. Radiochemical techniques were developed to make quantitative studies of the adsorption, an important part of these being practical procedures for obtaining sufficient cleanliness and freedeom from airborne contamination. Lack of uniformity of adsorption, as shown by autoradiography, was taken to indicate contamination, and techniques were developed to avid this. Other techniques enabled the continuous monitoring of the sample during adsorption or desorption kinetic experiments. It was shown that adsorption of phosphate on the untreated mica sheets was low, but the adsorption was greatly enhanced if the mica had been treated with aqueous solutions of certain cations such as gallium, aluminium and iron. Form the measurement of the amount of phosphate adsorbed, as a function of the conditions of aluminium treatment, it was concluded that the phosphate could be absorbed by at least three different processes, all of which could be of importance in phosphate fixation by soils. As well as these processes, which occurred on clean, flat, mica surfaces, there were others, involving the edges of mica and sheets, and unknown, but probably organic, films on both mica and air-water surfaces. These could all be of comparable importance in soils. The kinetic measurements of phosphate adsorption and desorption on aluminium-treated mica indicated that many hours were required for attainment of equilibrium, and were quantitatively consistent with the hypothesis that in some cases the adsorption and desorption kinetics were controlled by diffusion of phosphate into particles of some material, possibly a hydrous oxide, adsorbed on the mica. The existence of such particles was supported by the fact that up to one phosphate molecule per two square Angstrom units of mica surface was adsorbed, (and this did not appear to be a value at which the surface was saturated.) Kinetic measurements of 67 Ga sorption processes were consistent with diffusion of gallium through a thin water film, with a diffusion coefficient several orders of magnitude lower than that of single ions in free solution. This may indicate that the gallium was adsorbing as particles, in agreement with the requirements of the phosphate experiments.</p>

The Adsorption of Phosphate on Mica Surfaces

<p>The surface chemistry of the 001 face of cleaved mica sheets was studied with a view to understanding some of the fundamental processes underlying the phenomenon of fixation of phosphate by soils. Radiochemical techniques were developed to make quantitative studies of the adsorption, an important part of these being practical procedures for obtaining sufficient cleanliness and freedeom from airborne contamination. Lack of uniformity of adsorption, as shown by autoradiography, was taken to indicate contamination, and techniques were developed to avid this. Other techniques enabled the continuous monitoring of the sample during adsorption or desorption kinetic experiments. It was shown that adsorption of phosphate on the untreated mica sheets was low, but the adsorption was greatly enhanced if the mica had been treated with aqueous solutions of certain cations such as gallium, aluminium and iron. Form the measurement of the amount of phosphate adsorbed, as a function of the conditions of aluminium treatment, it was concluded that the phosphate could be absorbed by at least three different processes, all of which could be of importance in phosphate fixation by soils. As well as these processes, which occurred on clean, flat, mica surfaces, there were others, involving the edges of mica and sheets, and unknown, but probably organic, films on both mica and air-water surfaces. These could all be of comparable importance in soils. The kinetic measurements of phosphate adsorption and desorption on aluminium-treated mica indicated that many hours were required for attainment of equilibrium, and were quantitatively consistent with the hypothesis that in some cases the adsorption and desorption kinetics were controlled by diffusion of phosphate into particles of some material, possibly a hydrous oxide, adsorbed on the mica. The existence of such particles was supported by the fact that up to one phosphate molecule per two square Angstrom units of mica surface was adsorbed, (and this did not appear to be a value at which the surface was saturated.) Kinetic measurements of 67 Ga sorption processes were consistent with diffusion of gallium through a thin water film, with a diffusion coefficient several orders of magnitude lower than that of single ions in free solution. This may indicate that the gallium was adsorbing as particles, in agreement with the requirements of the phosphate experiments.</p>

Universal promotion of heterogeneous olefin metathesis catalysts by controlling dynamic active site renewal

Olefin metathesis is a versatile strategy for large-scale olefin interconversion, yet mechanistic details over industrial heterogeneous catalysts have remained ambiguous for decades. Here, from rigorous kinetic measurements, spectroscopic studies, and computational modeling of propylene metathesis over model and industrial WOx/SiO2 catalysts, we identify a hitherto unknown site renewal and decay cycle, mediated by proton transfers involving proximal Brønsted acidic OH groups, which operates concurrently with the classical Chauvin cycle. We show how this cycle can be manipulated using small quantities of promoter olefins to drastically increase steady-state propylene metathesis rates by up to 30-fold at 250oC with negligible promoter consumption. The increase in activity and considerable reduction of operating temperature requirements were also observed on MoOx/SiO2 catalysts, showing that this strategy is general and can address major roadblocks associated with industrial metathesis processes.

Effects of Knee Injury Length on Jump Inside Kick Performances of Wushu Player

Background and Objectives: When performing the jump inside kick in Wushu, it is important to understand the rotation technique while in mid-air. This is because the score varies according to the mid-air rotation, and when landing after the mid-air rotation, it causes considerable injury to the knee. This study aimed to compare the differences in kinematic and kinetic variables between experienced and less experienced knee injuries in the Wushu players who perform 360°, 540°, and 720° jump inside kicks in self-taolu. Materials and Methods: The participants’ mean (SD) age was 26.12 (2.84) years old. All of them had suffered knee injuries and were all recovering and returning to training. The group was classified into a group with less than 20 months of injury experience (LESS IG, n = 6) and a group with more than 20 months of injury experience (MORE IG, n = 6). For kinematic measurements, jump inside kicks at three rotations were assessed by using high-speed cameras. For kinetic measurements, the contraction time and maximal displacement of tensiomyography were assessed in the vastus lateralis, vastus medialis, rectus femoris, biceps femoris, gastrocnemius lateralis, gastrocnemius medialis, and tibialis anterior. The peak torque, work per repetition, fatigue index, and total work of isokinetic moments were assessed using knee extension/flexion, ankle inversion/eversion, and ankle plantarflexion/dorsiflexion tests. Results: Although there was no difference at the low difficulty level (360°), there were significant differences at the higher difficulty levels (540° and 720°) between the LESS IG and the MORE IG. For distance and time, the LESS IG had a shorter jump distance, but a faster rotation time compared to those in the MORE IG. Due to the characteristics of the jump inside kick’s rotation to the left, the static and dynamic muscle contractility properties were mainly found to be higher in the left lower extremity than in the right lower extremity, and higher in the LESS IG than in the MORE IG. In addition, this study observed that the ankle plantarflexor in the LESS IG was significantly higher than that in the MORE IG. Conclusion: To become a world-class self-taolu athlete while avoiding knee injuries, it is necessary to develop the static and dynamic myofunctions of the lower extremities required for jumping. Moreover, it is considered desirable to train by focusing on the vertical height and the amount of rotation during jumping.

Dietary protein requirements and recommendations for healthy older adults – A critical narrative review of the scientific evidence

Adequate protein intake is essential for the maintenance of whole-body protein mass. Different methodological approaches are used to substantiate the evidence for the current protein recommendation, and it is continuously debated whether older adults require more protein to counteract the age-dependent loss of muscle mass, sarcopenia. Thus, the purpose of this critical narrative review is to outline and discuss differences in the approaches and methodologies assessing the protein requirements and hence, resulting in controversies in current protein recommendations for healthy older adults. Through a literature search, this narrative review first summarizes the historical development of the FAO/WHO/UNU setting of protein requirements and recommendations for healthy older adults. Hereafter, we describe the various types of studies (epidemiological studies and protein turnover kinetic measurements) and applied methodological approaches founding the basis and the different recommendations with focus on healthy older adults. Finally, we discuss important factors to be considered in future studies to create evidence for international agreement on protein requirements and recommendations for healthy older adults. We conclude by proposing future directions to determine “true” protein requirement and recommendation for healthy older adults.

Chemical decarboxylation kinetics and identification of amino acid standards by benchtop NMR spectroscopy

In this paper, we examined the competence of amino acids as standards for instrumental biochemical analysis. The chosen amino acids were first dissolved in various aquatic solutions and then measured in a benchtop NMR spectrometer, which is not a common choice in such analytical investigations. Analysis by mass spectrometry was used in addition. As part of these investigations, we examined and determined the stability of the amino acids ornithine, glutamic acid, alanine, glycine, proline, pyroglutamic acid, phenylalanine and trans-4-hydroxy-D-proline under critical basic and acidic pH conditions and under various other conditions. We observed that not all solutions of the amino acid standards remain stable under the given conditions and a chemical transformation takes place. Given our findings by mass spectroscopy, additional kinetic measurements were carried out with the benchtop NMR spectrometer. We discovered that pyroglutamic acid becomes unstable under basic conditions and decarboxylates to pyrrolidone.

Kinetics and Mechanism of Formation of 2,3-Disubstituted 1,4-Benzothiazines: One Pot DMSO Assisted Cyclocondensation of Bisanilino Disulphide and 1, 3-Dicarbonyls

The kinetics of DMSO (dimethylsulphoxide) assisted one pot cyclocondensation of bisanilino disulphide and 1,3-dicarbonyls has been investigated using spectral method. The kinetic measurements have been carried by varying media, temperature and concentration of the reactants. The assistance of DMSO is investigated. The order of reaction, effects of substituents with dicarbonyls on the rates and thermodynamic parameters has also been reported. First time reaction conditions are optimised for performing the cyclocondensation conveniently and rapidly. Probably mechanism has been proposed and order of reaction was determined on the basis of rate expression and was in agreement with second order rate equation, used for the determination of rate constants.