Revealing the SARS-CoV-2 Spike Protein and Specific Antibody Immune Complex Formation Mechanism for Precise Evaluation of Antibody Affinity.

The profound understanding and detailed evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (SCoV2-S) protein and specific antibody interaction mechanism is of high importance in the development of immunosensors for COVID-19. In the present work, we studied a model system of immobilized SCoV2-S protein and specific monoclonal antibodies by molecular dynamics of immune complex formation in real time. We simultaneously applied spectroscopic ellipsometry and quartz crystal microbalance with dissipation to reveal the features and steps of the immune complex formation. We showed direct experimental evidence based on acoustic and optical measurements that the immune complex between covalently immobilized SCoV2-S and specific monoclonal antibodies is formed in two stages. Based on these findings it was demonstrated that applying a two-step binding mathematical model for kinetics analysis leads to a more precise determination of interaction rate constants than that determined by the 1:1 Langmuir binding model. Our investigation showed that the equilibrium dissociation constants (KD) determined by a two-step binding model and the 1:1 Langmuir model could differ significantly. The reported findings can facilitate a deeper understanding of antigen-antibody immune complex formation steps and can open a new way for the evaluation of antibody affinity towards corresponding antigens.

Investigation of SARS-CoV-2 nucleocapsid protein interaction with a specific antibody by combined spectroscopic ellipsometry and quartz crystal microbalance with dissipation.

Detailed evaluations of the antigen and antibody interaction rate and strength of the immune complex formed are very important for medical and bioanalytical applications. These data are crucial for the development of sensitive and fast immunosensors suitable for continuous measurements. Therefore, combined spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation (QCM-D) technique (SE/QCM-D) was used for the evaluation: (i)of covalent immobilization of SARS-CoV-2 nucleocapsid protein (SCoV2-N) on QCM-D sensor disc modified by self-assembled monolayer based on 11-mercaptoundecanoic acid and (ii)interaction of immobilized SCoV2-N with specific polyclonal anti-SCoV2-N antibodies followed by immune complex formation process. The results show that the SCoV2-N monolayer is rigid due to the low energy dissipation registered during the QCM-D measurement. In contrast, the anti-SCoV2-N layer produced after interaction with the immobilized SCoV2-N formed a soft and viscous layer. It was determined, that the sparse distribution of SCoV2-N on the surface affected the spatial arrangement of the antibody during the formation of immune complexes. The hinge-mediated flexibility of the antibody Fab fragments allows them to reach the more distantly located SCoV2-N and establish a bivalent binding between proteins in the formed SCoV2-N/anti-SCoV2-N complex. It was noted that the SE/QCM-D method can provide more precise quantitative information about the flexibility and conformational changes of antibody during the formation of the immune complex on the surface over time.

Investigation and Comparison of Specific Antibodies' Affinity Interaction with SARS-CoV-2 Wild-Type, B.1.1.7, and B.1.351 Spike Protein by Total Internal Reflection Ellipsometry.

SARS-CoV-2 vaccines provide strong protection against COVID-19. However, the emergence of SARS-CoV-2 variants has raised concerns about the efficacy of vaccines. In this study, we investigated the interactions of specific polyclonal human antibodies (pAb-SCoV2-S) produced after vaccination with the Vaxzevria vaccine with the spike proteins of three SARS-CoV-2 variants of concern: wild-type, B.1.1.7, and B.1.351. Highly sensitive, label-free, and real-time monitoring of these interactions was accomplished using the total internal reflection ellipsometry method. Thermodynamic parameters such as association and dissociation rate constants, the stable immune complex formation rate constant (kr), the equilibrium association and dissociation (KD) constants and steric factors (Ps) were calculated using a two-step irreversible binding mathematical model. The results obtained show that the KD values for the specific antibody interactions with all three types of spike protein are in the same nanomolar range. The KD values for B.1.1.7 and B.1.351 suggest that the antibody produced after vaccination can successfully protect the population from the alpha (B.1.1.7) and beta (B.1.351) SARS-CoV-2 mutations. The steric factors (Ps) obtained for all three types of spike proteins showed a 100-fold lower requirement for the formation of an immune complex when compared with nucleocapsid protein.

Nanostructured Manganite Films Grown by Pulsed Injection MOCVD: Tuning Low- and High-Field Magnetoresistive Properties for Sensors Applications.

The results of colossal magnetoresistance (CMR) properties of La0.83Sr0.17Mn1.21O3 (LSMO) films grown by pulsed injection MOCVD technique onto various substrates are presented. The films with thicknesses of 360 nm and 60 nm grown on AT-cut single crystal quartz, polycrystalline Al2O3, and amorphous Si/SiO2 substrates were nanostructured with column-shaped crystallites spread perpendicular to the film plane. It was found that morphology, microstructure, and magnetoresistive properties of the films strongly depend on the substrate used. The low-field MR at low temperatures (25 K) showed twice higher values (-31% at 0.7 T) for LSMO/quartz in comparison to films grown on the other substrates (-15%). This value is high in comparison to results published in literature for manganite films prepared without additional insulating oxides. The high-field MR measured up to 20 T at 80 K was also the highest for LSMO/quartz films (-56%) and demonstrated the highest sensitivity S = 0.28 V/T at B = 0.25 T (voltage supply 2.5 V), which is promising for magnetic sensor applications. It was demonstrated that Mn excess Mn/(La + Sr) = 1.21 increases the metal-insulator transition temperature of the films up to 285 K, allowing the increase in the operation temperature of magnetic sensors up to 363 K. These results allow us to fabricate CMR sensors with predetermined parameters in a wide range of magnetic fields and temperatures.

The Application of a CMR-B-Scalar Sensor for the Investigation of the Electromagnetic Acceleration of Type II Superconductors.

In this paper, we investigated the behavior of a type II superconducting armature when accelerated by a pulsed magnetic field generated by a single-stage pancake coil. While conducting this investigation, we performed a numerical finite element simulation and an experimental study of the magnetic field dynamics at the edge of the pancake coil when the payload was a superconducting disc made from YBa2Cu3O7-x, cooled down to 77 K. The magnetic field measurements were performed using a CMR-B-scalar sensor, which was able to measure the absolute magnitude of the magnetic field and was specifically manufactured in order to increase the sensor's sensitivity up to 500 mT. It was obtained that type II superconducting armatures can outperform normal metals when the launch conditions are tailored to their electromagnetic properties.

Magnetic Field Measurements during Magnetic Pulse Welding Using CMR-B-Scalar Sensors.

The possibility of applying CMR-B-scalar sensors made from thin manganite films exhibiting the colossal magnetoresistance effect as a fast-nondestructive method for the evaluation of the quality of the magnetic pulse welding (MPW) process is investigated in this paper. This method based on magnetic field magnitude measurements in the vicinity of the tools and joining parts was tested during the electromagnetic compression and MPW of an aluminum flyer tube with a steel parent. The testing setup used for the investigation allowed the simultaneous measurement of the flyer displacement, its velocity, and the magnitude of the magnetic field close to the flyer. The experimental results and simulations showed that, during the welding of the aluminum tube with the steel parent, the maximum magnetic field in the gap between the field shaper and the flyer is achieved much earlier than the maximum of the current pulse of the coil and that the first half-wave pulse of the magnetic field has two peaks. It was also found that the time instant of the minimum between these peaks depends on the charging energy of the capacitors and is associated with the collision of the flyer with the parent. Together with the first peak maximum and its time-position, this characteristic could be an indication of the welding quality. These results were confirmed by simultaneous measurements of the flyer displacement and velocity, as well as a numerical simulation of the magnetic field dynamics. The relationship between the peculiarities of the magnetic field pulse and the quality of the welding process is discussed. It was demonstrated that the proposed method of magnetic field measurement during magnetic pulse welding in combination with subsequent peel testing could be used as a nondestructive method for the monitoring of the quality of the welding process.

Evaluation of affinity sensor response kinetics towards dimeric ligands linked with spacers of different rigidity: Immobilized recombinant granulocyte colony-stimulating factor based synthetic receptor binding with genetically engineered dimeric analyte derivatives.

The modelling of protein-protein binding kinetics is important for the development of affinity-sensors and the prediction of signaling protein based drug efficiency. Therefore, in this research we have evaluated the binding kinetics of several genetically designed protein models: (i) three different ligands based on granulocyte colony-stimulating factor GCSF homo-dimeric derivatives linked by differed by linkers of different length and flexibility; (ii) an antibody-like receptor (GCSF-R) based on two GCSF-receptor sites immobilized to Fc domains, which are common parts of protein structures forming antibodies. Genetically engineered GCSF-R is similar to an antibody because it, like the antibody, has two binding sites, which both selectively bind with GCSF ligands. To design the affinity sensor model studied here, GCSF-R was immobilized on a thin gold layer via self-assembled monolayer conjugated with Protein-G. Binding kinetics between immobilized GCSF-R and all three different recombinant GCSF-based homo-dimeric derivatives were evaluated by total internal reflection ellipsometry. Association constants were determined by fitting mathematical models to the experimental data. It was clearly observed that both (i) affinity and (ii) binding kinetics depend on the length and flexibility of the linker that connects both domains of a GCSF-based ligand. The fastest association between immobilized GCSF-R and GCSF-based ligands was observed for ligands whose GCSF domains were interconnected by the longest and the most flexible linker. Here we present ellipsometry-based measurements and models of the interaction kinetics that advance the understanding of bidentate-receptor-based immunosensor action and enables us to predict the optimal linker structure for the design of GCSF-based medications.

The link between yeast cell wall porosity and plasma membrane permeability after PEF treatment.

An investigation of the yeast cell resealing process was performed by studying the absorption of the tetraphenylphosphonium (TPP+) ion by the yeast Saccharomyces cerevisiae. It was shown that the main barrier for the uptake of such TPP+ ions is the cell wall. An increased rate of TPP+ absorption after treatment of such cells with a pulsed electric field (PEF) was observed only in intact cells, but not in spheroplasts. The investigation of the uptake of TPP+ in PEF treated cells exposed to TPP+ for different time intervals also showed the dependence of the absorption rate on the PEF strength. The modelling of the TPP+ uptake recovery has also shown that the characteristic decay time of the non-equilibrium (PEF induced) pores was approximately a few tens of seconds and this did not depend on the PEF strength. A further investigation of such cell membrane recovery process using a florescent SYTOX Green nucleic acid stain dye also showed that such membrane resealing takes place over a time that is like that occurring in the cell wall. It was thus concluded that the similar characteristic lifetimes of the non-equilibrium pores in the cell wall and membrane after exposure  to  PEF indicate a strong coupling between these parts of the cell.

Room temperature Co-doped manganite/graphene sensor operating at high pulsed magnetic fields.

The demand to increase the sensitivity to magnetic field in a broad magnetic field ranges has led to the research of novel materials for sensor applications. Therefore, the hybrid system consisting of two different magnetoresistive materials - nanostructured Co-doped manganite La1-xSrx(Mn1-yCoy)zO3 and single- and few-layer graphene - were combined and investigated as potential system for magnetic field sensing. The negative colossal magnetoresistance (CMR) of manganite-cobaltite and positive one of graphene gives the possibility to increase the sensitivity to magnetic field of the hybrid sensor. The performed magnetoresistance (MR) measurements of individual few layer (n = 1-5) graphene structures revealed the highest MR values for three-layer graphene (3LG), whereas additional Co-doping increased the MR values of nanostructured manganite films. The connection of 3LG graphene and Co-doped magnanite film in a voltage divider configuration significantly increased the sensitivity of the hybrid sensor at low and intermediate magnetic fields (1-2 T): 70 mV/VT of hybrid sensor in comparison with 56 mV/VT for 3LG and 12 mV/VT for Co-doped magnanite film, respectively, and broadened the magnetic field operation range (0.1-20) T of the produced sensor prototype.

Hybrid graphene-manganite thin film structure for magnetoresistive sensor application.

An increasing demand of magnetic field sensors with high sensitivity at room temperatures and spatial resolution at micro-nanoscales has resulted in numerous investigations of physical phenomena in advanced materials, and fabrication of novel magnetoresistive devices. In this study the novel magnetic field sensor based on combination of a single layer graphene (SLG) and thin nanostructured manganite La0.8Sr0.2MnO3 (LSMO) film-hybrid graphene-manganite (GM) structure, is proposed and fabricated. The hybrid GM structure employs the properties of two materials-SLG and LSMO-on the nanoscale level and results in the enhanced sensitivity to magnetic field of the hybrid sensor on the macroscopic level. Such result is achieved by designing the hybrid GM sensor in a Wheatstone half-bridge which enables to employ in the device operation two effects of nanomaterials-large Lorentz force induced positive magnetoresistance of graphene and colossal negative magnetoresistance of nanostructured manganite film, and significantly increase the sensitivity S of the hybrid GM sensor in comparison with the individual SLG and LSMO sensors: S = 5.5 mV T-1 for SLG, 14.5 mV T-1 for LSMO and 20 mV T-1 for hybrid GM at 0.5 T, when supply voltage was 1.249 V. The hybrid GM sensor operates in the range of (0.1-2.3) T and has lower sensitivity to temperature variations in comparison to the manganite sensor. Moreover, it can be applied for position sensing. The ability to control sensor's characteristics by changing technological conditions of the fabrication of hybrid structure and tuning the nanostructure properties of manganite film is discussed.

Electric field-induced effects on yeast cell wall permeabilization.

The permeability of the yeast cells (Saccharomyces cerevisiae) to lipophilic tetraphenylphosphonium cations (TPP(+) ) after their treatment with single square-shaped strong electric field pulses was analyzed. Pulsed electric fields (PEF) with durations from 5 to 150 µs and strengths from 0 to 10 kV/cm were applied to a standard electroporation cuvette filled with the appropriate buffer. The TPP(+) absorption process was analyzed using an ion selective microelectrode (ISE) and the plasma membrane permeability was determined by measurements obtained using a calcein blue dye release assay. The viability of the yeast and the inactivation of the cells were determined using the optical absorbance method. The experimental data taken after yeasts were treated with PEF and incubated for 3 min showed an increased uptake of TPP(+) by the yeast. This process can be controlled by setting the amplitude and pulse duration of the applied PEF. The kinetics of the TPP(+) absorption process is described using the second order absolute rate equation. It was concluded that the changes of the charge on the yeast cell wall, which is the main barrier for TPP(+) , is due to the poration of the plasma membrane. The applicability of the TPP(+) absorption measurements for the analysis of yeast cells electroporation process is also discussed.