Establishing Quartz Crystal Microbalance with Dissipation (QCM-D) Coupled with Spectroscopic Ellipsometry (SE) as an Advantageous Technique for the Characterization of Ultra-Thin Film Hydrogels.

Despite the considerable significance of utilizing ultra-thin film (utf) hydrogels as multipurpose platforms for biomedical applications, there is still an important lack of adequate characterization techniques suitable for such materials. In this Perspective, the use of quartz crystal microbalance with dissipation (QCM-D) coupled with spectral ellipsometry (SE) is presented as a potential tool for the complete characterization of utf-hydrogels due to its nanometric sensitivity and high versatility. Herein, the fundaments for utf-hydrogel characterization are settled down as far as the QCM-D/SE response is explored under a wide range of different in operando wet working conditions measurements such as temperature or liquid media, among others. Therefore, the design of measuring protocols capable to perform is proposed and compiled, for the first time, complete and precise characterization of the cross-link density, thickness variations (swelling ratio determination), stability analyses, and mechanical studies (including the simultaneous generation of stress-strain curves and the evaluation of the viscoelastic; leading to the final determination of the Poisson's ratio) under different in operando conditions. Finally, the future challenges and implications for the general characterization of soft-thin films are discussed.

Illuminating Pathways to Dynamic Nanotribology: Light-Mediated Active Control of Interfacial Friction with Nanosuspensions.

Active control of nanotribological properties is a challenge. Materials responsive to external stimuli may catalyze this paradigm shift. Recently, the nanofriction of a thin film is modulated by light, ushering in phototribology. This frontier is expanded here, by investigating photoactive nanoparticles in lubricants to confer similar functionality to passive surfaces. Quartz-crystal microbalance (QCM) is employed to assess the phototribological behavior of aqueous suspensions of titanium dioxide nanoparticles. A comparison of dark and illuminated conditions provides the first demonstration of tuning the interfacial friction in solid-nanosuspension interfaces by light. Cyclic tests reveal reversible transitions between higher (dark) and lower friction (illuminated) regimes. These transitions are underpinned by transient states with surface charge variations, as confirmed by Zeta potential measurements. The accumulated surface charge increases repulsion within the system and favors sliding. Upon cessation of illumination, the system returns to its prior equilibrium state. These findings impact not only nanotribology but nanofluidics and nanorheology. Furthermore, the results underscore the need to consider light-induced effects in other scenarios, including the calculation of activity coefficients of photoactive suspensions. This multifaceted study introduces a new dimension to in operando frictional tuning, beckoning a myriad of applications and fundamental insights at the nanoscale.

Design and development of a portable low-cost QCM-based system for liquid biosensing.

Quartz crystal microbalance (QCM) is a versatile sensing platform that has gained increasing attention for its use in bioapplications due to its high sensitivity, real-time measurement capabilities, and label-free detection. This article presents a portable QCM system for liquid biosensing that uses a modified Hartley oscillator to drive 14 mm-diameter commercial QCM sensors. The system is designed to be low-cost, easy to use, and highly sensitive, making it ideal for various bioapplications. A new flow cell design to deliver samples to the surface of the sensor has been designed, fabricated, and tested. For portability and miniaturization purposes, a micropump-based pumping system is used in the current system. The system has a built-in temperature controller allowing for accurate frequency measurements. In addition, the system can be used in benchtop mode. The capability of the present system to be used in liquid biosensing is demonstrated through an experimental test for sensitivity to changes in the viscosity of glycerol samples. It was found to have a sensitivity of 263.51 Hz/mPa.s using a 10 MHz QCM sensor. Future work regarding potential applications was suggested.

Alternative strategies for the recombinant synthesis, DOPA modification and analysis of mussel foot proteins - A case study for Mefp-3 from Mytilus edulis.

Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus edulis foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble and tag-free Mefp-3 using the SUMO-fusion technology. Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOF-MS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification.

Nanoscale Insights into the Interaction Mechanism Underlying the Adsorption and Retention of Heavy Metal Ions by Humic Acid.

The mobility and distribution of heavy metal ions (HMs) in aquatic environments are significantly influenced by humic acid (HA), which is ubiquitous. A quantitative understanding of the interaction mechanism underlying the adsorption and retention of HMs by HA is of vital significance but remains elusive. Herein, the interaction mechanism between HA and different types of HMs (i.e., Cd(II), Pb(II), arsenate, and chromate) was quantitatively investigated at the nanoscale. Based on quartz crystal microbalance with dissipation tests, the adsorption capacities of Pb(II), Cd(II), As(V), and Cr(VI) ionic species on the HA surface were measured as ∼0.40, ∼0.25, ∼0.12, and ∼0.02 nmol cm-2, respectively. Atomic force microscopy force results showed that the presence of Pb(II)/Cd(II) cations suppressed the electrostatic double-layer repulsion during the approach of two HA surfaces and the adhesion energy during separation was considerably enhanced from ∼2.18 to ∼5.05/∼4.18 mJ m-2. Such strong adhesion stems from the synergistic metal-HA complexation and cation-π interaction, as evidenced by spectroscopic analysis and theoretical simulation. In contrast, As(V)/Cr(VI) oxo-anions could form only weak hydrogen bonds with HA, resulting in similar adhesion energies for HA-HA (∼2.18 mJ m-2) and HA-As(V)/Cr(VI)-HA systems (∼2.26/∼1.96 mJ m-2). This work provides nanoscale insights into quantitative HM-HA interactions, improving the understanding of HMs biogeochemical cycling.

Real-world evaluation of a QCM-based biosensor for exhaled air.

The biosensor, named "virusmeter" in this study, integrates quartz crystal microbalance technology with an immune-functionalized chip to distinguish between symptomatic patients with respiratory diseases and healthy individuals by analyzing exhaled air samples. Renowned for its compact design, rapidity, and noninvasive nature, this device yields results within a 5-min timeframe. Evaluated under controlled conditions with 54 hospitalized symptomatic COVID-19 patients and 128 control subjects, the biosensor demonstrated good overall sensitivity (98.15%, 95% CI 90.1-100.0) and specificity (96.87%, 95% CI 92.2-99.1). This proof-of-concept presents an innovative approach with significant potential for leveraging piezoelectric sensors to diagnose respiratory diseases.

Thickness Dependent CO2 Adsorption of Poly(ethyleneimine) Thin Films for Direct Air Capture.

Mesoporous silica impregnated with polyethyleneimine (PEI) has been shown to be a suitable material for the direct air capture (DAC) of CO2. Factors such as CO2 concentration, temperature, and amine loading impact overall capture capacity and amine efficiency by altering diffusional resistance and reaction kinetics. When studied in the impregnated 3-dimensional sorbent material, internal diffusion impacts the evaluation of the reaction kinetics at the air/amine interface. In this work, we designed a novel tandem quartz crystal microbalance with dissipation (QCM-D) and polarization modulation infrared reflective absorption spectroscopy (PM-IRRAS) instrument. CO2 adsorption kinetics of the PEI-based amine layer in a 2-dimensional geometry were studied at a variety of film thicknesses (10 nm to 100 nm), temperatures (25 °C to 80 °C), and CO2 concentrations (5 % and 0.04 % by mole fraction). Total CO2 capture capacity increased with film thickness but decreased amine efficiency, as additional diffusional resistance for thicker films limits access to available amine sites. The capture capacity of thick films (>50 nm) is shown to be limited by amine availability, while capture of thin films (<50 nm) is limited by CO2 availability. A 50 nm PEI film was shown to be optimal for capture of 0.04 % (400 ppm) CO2. The adsorption profiles for these conditions were fitted to pseudo-first order and Avrami fractional order models. The reaction process switches between a diffusion limited reaction to a kinetic limited reaction at 80 °C when using 5 % CO2 and 55 °C when using 0.04 % CO2. These results offer accurate analysis of adsorption of CO2 at the air/amine interface of PEI films which can be used for the design of future sorbent materials.

Identification of medium- and mechanism-related pitfalls towards improved performance and applicability of electrochemical mercury(II) aptasensors.

The importance of understanding the mercury (II) ion interactions with thymine-rich DNA sequences is the reason for multiple comparative investigations carried out with the use of optical detection techniques directly in the depth of solution. However, the results of such investigations have limited applicability in the interpretation of the Hg2+ binding phenomenon by DNA sequences in thin, interfacial (electrode/solution), self-organized monolayers immobilized on polarizable surfaces, often used for sensing purposes in electrochemical biosensors. Overlooking the careful optimization of the measurement conditions is the source of discrepancies in the interpretation of the registered electrochemical signal. In this study, the chosen effects accompanying the efficiency of surface related recognition of Hg2+ by polyThymine DNA sequences labelled with methylene blue were investigated by voltammetry, QCM and spectro-electrochemical techniques. As was shown, the composition of the biosensing layer and buffers or the analytical procedures have a significant impact on the registered electrochemical readout which translates into signal stability, the biosensor's working parameters or even the mechanism of detection. After elucidation of the above factors, the complete and ready-to-use biosensor-based analytical solution was proposed offering subpicomolar mercury ion determination with high selectivity (also in aqueous real samples), reusability, and high signal stability even after long-term storage. The developed procedures were successfully used during the miniaturization process with self-prepared (PVD) elastic transducers. The obtained sensor, together with the simplicity of its use, low manufacturing cost, and attractive analytical parameters (i.e., LOD < < Hg2+ WHO limit) can present an interesting alternative for on-site mercury ion detection in environmental samples.

Improving Aerosol Characterization Using an Optical Particle Counter Coupled with a Quartz Crystal Microbalance with an Integrated Microheater.

Aerosols, as well as suspended particulate matter, impact atmospheric pollution, the climate, and human health, directly or indirectly. Particle size, chemical composition, and other aerosol characteristics are determinant factors for atmospheric pollution dynamics and more. In the last decade, low-cost devices have been widely used in instrumentation to measure aerosols. However, they present some issues, such as the problem of discriminating whether the aerosol is composed of liquid particles or solid. This issue could lead to errors in the estimation of mass concentration in monitoring environments where there is fog. In this study, we investigate the use of an optical particle counter (OPC) coupled to a quartz crystal microbalance with an integrated microheater (H-QCM) to enhance measurement performances. The H-QCM was used not only to measure the collected mass on its surface but also, by using the integrated microheater, it was able to heat the collected mass by performing heating cycles. In particular, we tested the developed system with aerosolized saline solutions of sodium chloride (NaCl), with three decreasing concentrations of salt and three electronic cigarette solutions (e-liquid), with different concentrations of propylene glycol and glycerin mixtures. The results showed that the OPC coherently counted the salt dilution effects, and the H-QCM output confirmed the presence of liquid and solid particles in the aerosols. In the case of e-liquid aerosols, the OPC counted the particles, and the HQCM output highlighted that in the aerosol, there were no solid particles but a liquid phase only. These findings contribute to the refinement of aerosol measurement methodologies by low-cost sensors, fostering a more comprehensive understanding.

Hydrogen-Bond Acidic Materials in Acoustic Wave Sensors for Nerve Chemical Warfare Agents' Detection.

The latest trends in the field of the on-site detection of chemical warfare agents (CWAs) involve increasing the availability of point detectors to enhance the operational awareness of commanders and soldiers. Among the intensively developed concepts aimed at meeting these requirements, wearable detectors, gas analyzers as equipment for micro- and mini-class unmanned aerial vehicles (UAVs), and distributed sensor networks can be mentioned. One of the analytical techniques well suited for use in this field is surface acoustic wave sensors, which can be utilized to construct lightweight, inexpensive, and undemanding gas analyzers for detecting CWAs. This review focuses on the intensively researched and developed variant of this technique, utilizing absorptive sensor layers dedicated for nerve CWAs' detection. The paper describes the mechanism of the specific interaction occurring between the target analyte and the sensing layer, which serves as the foundation for their selective detection. The main section of this paper includes a chronological review of individual achievements in the field, largely based on the peer-reviewed scientific literature dating back to the mid-1980s to the present day. The final section presents conclusions regarding the prospects for the development of this analytical technique in the targeted application.

New Insights into Aptamers: An Alternative to Antibodies in the Detection of Molecular Biomarkers.

Aptamers are short oligonucleotides with single-stranded regions or peptides that recently started to transform the field of diagnostics. Their unique ability to bind to specific target molecules with high affinity and specificity is at least comparable to many traditional biorecognition elements. Aptamers are synthetically produced, with a compact size that facilitates deeper tissue penetration and improved cellular targeting. Furthermore, they can be easily modified with various labels or functional groups, tailoring them for diverse applications. Even more uniquely, aptamers can be regenerated after use, making aptasensors a cost-effective and sustainable alternative compared to disposable biosensors. This review delves into the inherent properties of aptamers that make them advantageous in established diagnostic methods. Furthermore, we will examine some of the limitations of aptamers, such as the need to engage in bioinformatics procedures in order to understand the relationship between the structure of the aptamer and its binding abilities. The objective is to develop a targeted design for specific targets. We analyse the process of aptamer selection and design by exploring the current landscape of aptamer utilisation across various industries. Here, we illuminate the potential advantages and applications of aptamers in a range of diagnostic techniques, with a specific focus on quartz crystal microbalance (QCM) aptasensors and their integration into the well-established ELISA method. This review serves as a comprehensive resource, summarising the latest knowledge and applications of aptamers, particularly highlighting their potential to revolutionise diagnostic approaches.

The effect of probe density coverage on the detection of oenological tannins in quartz crystal microbalance with dissipation monitoring (QCM-D) experiments.

BACKGROUND: Polyphenols are a group of compounds found in grapes, musts, and wines. Their levels are crucial for grape ripening, proper must fermentation, and final wine characteristics. Standard chemical analysis is commonly used to detect these compounds, but it is costly, time consuming, and requires specialized laboratories and operators. To address this, this study explores a functionalized acoustic sensor for detecting oenological polyphenols. RESULTS: The method involves utilizing a quartz crystal microbalance with dissipation monitoring (QCM-D) to detect the target analyte by using a gelatin-based probe layer. The sensor is functionalized by optimizing the probe coverage density to maximize its performance. This is achieved by using 12-mercaptododecanoic acid (12-MCA) to immobilize the probe onto the gold sensor surface, and dithiothreitol (DTT) as a reducing and competitive binding agent. The concentration of 12-MCA and DTT in the solutions is varied to control the probe density. QCM-D measurements demonstrate that the probe density can be effectively adjusted using this approach, ranging from 0.2 × 1013 to 2 × 1013 molecules cm-2 . This study also investigates the interaction between the probe and tannins, confirming the ability of the sensor to detect them. Interestingly, the lower probe coverage achieves higher detection signals when normalized to probe immobilization signals. Moreover, significant changes in mechanical properties of the functionalization layer are observed after the interaction with samples. CONCLUSION: The combination of QCM-D with gelatin functionalization holds great promise for future applications in the wine industry. It offers real-time monitoring capabilities, requires minimal sample preparation, and provides high sensitivity for quality control purposes. © 2024 Society of Chemical Industry.

Cholesterol-Containing Liposomes Decorated With Au Nanoparticles as Minimal Tunable Fusion Machinery.

Membrane fusion is essential for the basal functionality of eukaryotic cells. In physiological conditions, fusion events are regulated by a wide range of specialized proteins, operating with finely tuned local lipid composition and ionic environment. Fusogenic proteins, assisted by membrane cholesterol and calcium ions, provide the mechanical energy necessary to achieve vesicle fusion in neuromediator release. Similar cooperative effects must be explored when considering synthetic approaches for controlled membrane fusion. We show that liposomes decorated with amphiphilic Au nanoparticles (AuLips) can act as minimal tunable fusion machinery. AuLips fusion is triggered by divalent ions, while the number of fusion events dramatically changes with, and can be finely tuned by, the liposome cholesterol content. We combine quartz-crystal-microbalance with dissipation monitoring (QCM-D), fluorescence assays, and small-angle X-ray scattering (SAXS) with molecular dynamics (MD) at coarse-grained (CG) resolution, revealing new mechanistic details on the fusogenic activity of amphiphilic Au nanoparticles (AuNPs) and demonstrating the ability of these synthetic nanomaterials to induce fusion regardless of the divalent ion used (Ca2+ or Mg2+ ). The results provide a novel contribution to developing new artificial fusogenic agents for next-generation biomedical applications that require tight control of the rate of fusion events (e.g., targeted drug delivery).

Eco-Corona Formation on Plastics: Adsorption of Dissolved Organic Matter to Pristine and Photochemically Weathered Polymer Surfaces.

Plastic fate in freshwater systems is dependent on particle size, morphology, and physicochemical surface properties (e.g., charge, surface roughness, and hydrophobicity). Environmental aging processes, such as photochemical weathering and eco-corona formation due to dissolved organic matter (DOM) adsorption on plastic surfaces, can alter their physicochemical properties, affecting fate and transport. While plastic aging has been studied from a materials science perspective, its specific implications in environmental contexts remain less understood. Although photochemical weathering and eco-corona formation occur simultaneously in the environment, in this work, we systematically assessed the effects of photochemical weathering on the physicochemical properties of polymers (polyethylene, polypropylene, polyethylene terephthalate, and polystyrene) and how this influences the adsorption of DOMs (Suwannee River humic acid, fulvic acid, and natural organic matter) relative to pristine polymers. Pristine polymers initially had different and distinct physicochemical surface properties, but upon aging, they became more similar in terms of surface properties. Photochemical weathering resulted in a decrease in polymer film thickness, an increase in surface roughness, and hydrophilicity. DOM adlayers on the polymer surfaces resulted in more comparable wettability, effectively masking the initial polymer properties. Collectively, this study explores the physiochemical changes polymers undergo in laboratory studies mimicking environmental conditions. Understanding these changes is the initial step to rationalizing and predicting processes and interactions such as heteroaggregation that dictate the fate of plastics in the environment.

Effects of Oscillation Amplitude Variations on QCM Response to Microspheres of Different Sizes.

Suspended particulate matter (PMx) is one of the most important environmental pollutants. Miniaturized sensors capable of measuring and analyzing PMx are crucial in environmental research fields. The quartz crystal microbalance (QCM) is one of the most well-known sensors that could be used to monitor PMx. In general, in environmental pollution science, PMx is divided into two main categories correlated to particle diameter (e.g., PM < 2.5 µm and PM < 10 µm). QCM-based systems are capable of measuring this range of particles, but there is an important issue that limits the application. In fact, if particles with different diameters are collected on QCM electrodes, the response will be a result of the total mass of particles; there are no simple methods to discriminate the mass of the two categories without the use of a filter or manipulation during sampling. The QCM response depends on particle dimensions, fundamental resonant frequency, the amplitude of oscillation, and system dissipation properties. In this paper, we study the effects of oscillation amplitude variations and fundamental frequency (10, 5, and 2.5 MHz) values on the response, when particle matter with different sizes (2 µm and 10 µm) is deposited on the electrodes. The results showed that the 10 MHz QCM was not capable of detecting the 10 µm particles, and its response was not influenced by oscillation amplitude. On the other hand, the 2.5 MHz QCM detected the diameters of both particles, but only if a low amplitude value was used.

Effect of Load on Quartz Crystal Microbalance Sensor Response Addressed Using Fractional Order Calculus.

To accurately model the effect of the load caused by a liquid medium as a function of its viscosity, the fractional order Butterworth-Van Dyke (BVD) model of the QCM sensor is proposed in this study. A comprehensive understanding of the fractional order BVD model followed by a simulation of situations commonly encountered in experimental investigations underpins the new QCM sensor approach. The Levenberg-Marquardt (LM) algorithm is used in two fitting steps to extract all parameters of the fractional order BVD model. The integer-order electrical parameters were determined in the first step and the fractional order parameters were extracted in the second step. A parametric investigation was performed in air, water, and glycerol-water solutions in ten-percent steps for the fractional order BVD model. This indicated a change in the behavior of the QCM sensor when it swapped from air to water, modeled by the fractional order BVD model, followed by a specific dependence with increasing viscosity of the glycerol-water solution. The effect of the liquid medium on the reactive motional circuit elements of the BVD model in terms of fractional order calculus (FOC) was experimentally demonstrated. The experimental results demonstrated the value of the fractional order BVD model for a better understanding of the interactions occurring at the QCM sensor surface.

Spurious Resonance of the QCM Sensor: Load Analysis Based on Impedance Spectroscopy.

A research topic of equal importance to technological and application fields related to quartz crystal is the presence of unwanted responses known as spurious resonances. Spurious resonances are influenced by the surface finish of the quartz crystal, its diameter and thickness, and the mounting technique. In this paper, spurious resonances associated with fundamental resonance are studied by impedance spectroscopy to determine their evolution under load conditions. Investigation of the response of these spurious resonances provides new insights into the dissipation process at the QCM sensor surface. The significant increase of the motional resistance for spurious resonances at the transition from air to pure water is a specific situation revealed experimentally in this study. It has been shown experimentally that in the range between the air and water media, spurious resonances are much more attenuated than the fundamental resonance, thus providing support for investigating the dissipation process in detail. In this range, there are many applications in the field of chemical sensors or biosensors, such as VOC sensors, humidity sensors, or dew point sensors. The evolution of D factor with increasing medium viscosity is significantly different for spurious resonances compared to fundamental resonance, suggesting the usefulness of monitoring them in liquid media.

Effect of Noise on Determining Ultrathin-Film Parameters from QCM-D Data with the Viscoelastic Model.

Quartz crystal microbalance with dissipation monitoring (QCM-D) is a well-established technique for studying soft films. It can provide gravimetric as well as nongravimetric information about a film, such as its thickness and mechanical properties. The interpretation of sets of overtone-normalized frequency shifts, ∆f/n, and overtone-normalized shifts in half-bandwidth, ΔΓ/n, provided by QCM-D relies on a model that, in general, contains five independent parameters that are needed to describe film thickness and frequency-dependent viscoelastic properties. Here, we examine how noise inherent in experimental data affects the determination of these parameters. There are certain conditions where noise prevents the reliable determination of film thickness and the loss tangent. On the other hand, we show that there are conditions where it is possible to determine all five parameters. We relate these conditions to the mathematical properties of the model in terms of simple conceptual diagrams that can help users understand the model's behavior. Finally, we present new open source software for QCM-D data analysis written in Python, PyQTM.

Evaluation of Linkers' Influence on Peptide-Based Piezoelectric Biosensors' Sensitivity to Aldehydes in the Gas Phase.

Recent findings qualified aldehydes as potential biomarkers for disease diagnosis. One of the possibilities is to use electrochemical biosensors in point-of-care (PoC), but these need further development to overcome some limitations. Currently, the primary goal is to enhance their metrological parameters in terms of sensitivity and selectivity. Previous findings indicate that peptide OBPP4 (KLLFDSLTDLKKKMSEC-NH2) is a promising candidate for further development of aldehyde-sensitive biosensors. To increase the affinity of a receptor layer to long-chain aldehydes, a structure stabilization of the peptide active site via the incorporation of different linkers was studied. Indeed, the incorporation of linkers improved sensitivity to and binding of aldehydes in comparison to that of the original peptide-based biosensor. The tendency to adopt disordered structures was diminished owing to the implementation of suitable linkers. Therefore, to improve the metrological characteristics of peptide-based piezoelectric biosensors, linkers were added at the C-terminus of OBPP4 peptide (KLLFDSLTDLKKKMSE-linker-C-NH2). Those linkers consist of proteinogenic amino acids from group one: glycine, L-proline, L-serine, and non proteinogenic amino acids from group two: ß-alanine, 4-aminobutyric acid, and 6-aminohexanoic acid. Linkers were evaluated with in silico studies, followed by experimental verification. All studied linkers enhanced the detection of aldehydes in the gas phase. The highest difference in frequency (60 Hz, nonanal) was observed between original peptide-based biosensors and ones based on peptides modified with the GSGSGS linker. It allowed evaluation of the limit of detection for nonanal at the level of 2 ppm, which is nine times lower than that of the original peptide. The highest sensitivity values were also obtained for the GSGSGS linker: 0.3312, 0.4281, and 0.4676 Hz/ppm for pentanal, octanal, and nonanal, respectively. An order of magnitude increase in sensitivity was observed for the six linkers used. Generally, the linker's rigidity and the number of amino acid residues are much more essential for biosensors' metrological characteristics than the amino acid sequence itself. It was found that the longer the linkers, the better the effect on docking efficiency.

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.