Quartz Crystal Microbalance Frequency Response to Discrete Adsorbates in Liquids.

Quartz crystal microbalance with dissipation monitoring (QCM-D) has become a major tool enabling accurate investigation of the adsorption kinetics of nanometric objects such as DNA fragments, polypeptides, proteins, viruses, liposomes, polymer, and metal nanoparticles. However, in liquids, a quantitative analysis of the experimental results is often intricate because of the complex interplay of hydrodynamic and adhesion forces varying with the physicochemical properties of adsorbates and functionalized QCM-D sensors. In the present paper, we dissect the role of hydrodynamics for the analytically tractable case of stiff contact, whereas the adsorbed rigid particles oscillate with the resonator without rotation. Under the assumption of the low surface coverage, we theoretically study the excess shear force exerted on the resonator, which has two contributions: (i) the fluid-mediated force due to flow disturbance created by the particle and (ii) the force exerted on the particle by the fluid and transmitted to the sensor via contact. The theoretical analysis enables an accurate interpretation of the QCM-D impedance measurements. It is demonstrated inter alia that for particles of the size comparable with protein molecules, the hydrodynamic force dominates over the inertial force and that the apparent mass derived from QCM independently of the overtone is about 10 times the Sauerbrey (inertial) mass. The theoretical results show excellent agreement with the results of experiments and advanced numerical simulations for a wide range of particle sizes and oscillation frequencies.

Kinetics of Human Serum Albumin Adsorption on Polycation Functionalized Silica.

The adsorption kinetics of human serum albumin (HSA) on bare and poly-L-arginine (PARG)-modified silica substrates were investigated using reflectometry and atomic force microscopy (AFM). Measurements were carried out at various pHs, flow rates and albumin concentrations in the 10 and 150 mM NaCl solutions. The mass transfer rate constants and the maximum protein coverages were determined for the bare silica at pH 4.0 and theoretically interpreted in terms of the hybrid random sequential adsorption model. These results were used as reference data for the analysis of adsorption kinetics at larger pHs. It was shown that the adsorption on bare silica rapidly decreased with pH and became negligible at pH 7.4. The albumin adsorption on PARG-functionalized silica showed an opposite trend, i.e., it was negligible at pH 4 and attained maximum values at pH 7.4 and 150 mM NaCl, the conditions corresponding to the blood serum environment. These results were interpreted as the evidence of a significant role of electrostatic interactions in the albumin adsorption on the bare and PARG-modified silica. It was also argued that our results can serve as useful reference data enabling a proper interpretation of protein adsorption on substrates functionalized by polyelectrolytes.

Anisotropic Particle Deposition Kinetics from Quartz Crystal Microbalance Measurements: Beyond the Sphere Paradigm.

Deposition kinetics of polymer particles characterized by a prolate spheroid shape on gold sensors modified by the adsorption of poly(allylamine) was investigated using a quartz crystal microbalance and atomic force microscopy. Reference measurements were also performed for polymer particles of a spherical shape and the same diameter as the spheroid shorter axis. Primarily, the frequency and dissipation shifts for various overtones were measured as a function of time. These kinetic data were transformed into the dependence of the complex impedance, scaled up by the inertia impedance, upon the particle size to the hydrodynamic boundary layer ratio. The results obtained for low particle coverage were interpolated, which enabled the derivation of Sauerbrey-like equations, yielding the real particle coverage using the experimental frequency or dissipation (bandwidth) shifts. Experiments carried out for a long deposition time confirmed that, for spheroids, the imaginary and real impedance components were equal to each other for all overtones and for a large range of particle coverage. This result was explained in terms of a hydrodynamic, lubrication-like contact of particles with the sensor, enabling their sliding motion. In contrast, the experimental data obtained for spheres, where the impedance ratio was a complicated function of overtones and particle coverage, showed that the contact was rather stiff, preventing their motion over the sensor. It was concluded that results obtained in this work can be exploited as useful reference systems for a quantitative interpretation of bioparticle, especially bacteria, deposition kinetics on macroion-modified surfaces.

Streaming Current for Surfaces Covered by Square and Hexagonal Monolayers of Spherical Particles.

The interface and particle contributions to the streaming current of flat substrates covered with ordered square or hexagonal monolayers of spherical particles were theoretically evaluated for particle coverage up to close packing. The exact numerical results were approximated using fitting functions that contain exponential and linear terms to account for hydrodynamic screening and charge convection from the particle surfaces exposed to external flow. According to our calculations, the streaming currents for the ordered and random particle arrangements differ within a typical experimental error. Thus, streaming-current measurements, supplemented with our fitting functions, can be conveniently used to evaluate the particle coverage without detailed knowledge of the particle distribution. Our results for equal interface and particle ζ-potentials indicate that roughness can reduce the streaming current by more than 30%, even in the limit of the small size of spherical roughness asperities.

Mechanisms of Fibroblast Growth Factor 21 Adsorption on Macroion Layers: Molecular Dynamics Modeling and Kinetic Measurements.

Molecular dynamic modeling and various experimental techniques, including multi-angle dynamic light scattering (MADLS), streaming potential, optical waveguide light spectroscopy (OWLS), quartz crystal microbalance with dissipation (QCM), and atomic force microscopy (AFM), were applied to determine the basic physicochemical parameters of fibroblast growth factor 21 in electrolyte solutions. The protein size and shape, cross-section area, dependence of the nominal charge on pH, and isoelectric point of 5.3 were acquired. These data enabled the interpretation of the adsorption kinetics of FGF 21 on bare and macrocation-covered silica investigated by OWLS and QCM. It was confirmed that the protein molecules irreversibly adsorbed on the latter substrate, forming layers with controlled coverage up to 0.8 mg m-2, while their adsorption on bare silica was much smaller. The viability of two cell lines, CHO-K1 and L-929, on both bare and macrocation/FGF 21-covered substrates was also determined. It is postulated that the acquired results can serve as useful reference systems for designing complexes that can extend the half-life of FGF 21 in its active state.

Quantifying Nanoparticle Layer Topography: Theoretical Modeling and Atomic Force Microscopy Investigations.

A comprehensive method consisting of theoretical modeling and experimental atomic force microscopy (AFM) measurements was developed for the quantitative analysis of nanoparticle layer topography. Analytical results were derived for particles of various shapes such as cylinders (rods), disks, ellipsoids, hemispheres (caps), etc. It was shown that for all particles, their root-mean-square (rms) parameter exhibited a maximum at the coverage about 0.5, whereas the skewness was a monotonically decreasing function of the coverage. This enabled a facile determination of the particle coverage in the layer, even if the shape and size were not known. The validity of the analytical results was confirmed by computer modeling and experimental data acquired by AFM measurements for polymer nanoparticle deposition on mica and silica. The topographical analysis developed in this work can be exploited for a quantitative characterization of self-assembled layers of nano- and bioparticles, e.g., carbon nanotubes, silica and noble metal particles, DNA fragments, proteins, vesicles, viruses, and bacteria at solid surfaces. The acquired results also enabled a proper calibration, in particular the determination of the measurement precision, of various electron and scanning probe microscopies, such as AFM.

Synthesis and Characterization of Boron Carbide Nanoparticles as Potential Boron-Rich Therapeutic Carriers.

Boron carbide is one of the hardest materials in the world which can be synthesized by various methods. The most common one is a carbothermic or magnesiothermic reduction of B2O3 performed at high temperatures, where the obtained powder still requires grinding and purification. The goal of this research is to present the possibility of synthesizing B4C nanoparticles from elements via vapor deposition and modifying the morphology of the obtained powders, particularly those synthesized at high temperatures. B4C nanoparticles were synthesized in the process of direct synthesis from boron and carbon powders heated at the temperature of 1650 °C for 2 h under argon and characterized by using scanning electron microscopy, transmission electron microscopy, atomic force microscopy, X-ray diffraction analysis, and dynamic light scattering measurements. The physicochemical characteristics of B4C nanoparticles were determined, including the diffusion coefficients, hydrodynamic diameter, electrophoretic mobilities, and zeta potentials. An evaluation of the obtained B4C nanoparticles was performed on several human and mouse cell lines, showing the relation between the cytotoxicity effect and the size of the synthesized nanoparticles. Assessing the suitability of the synthesized B4C for further modifications in terms of its applicability in boron neutron capture therapy was the overarching goal of this research.

Mechanism of Anti-Salmonella Rabbit Immunoglobulin Adsorption on Polymer Particles.

The adsorption of anti-Salmonella rabbit immunoglobulin (IgaR) on negatively charged polymer particles leading to the formation of immunolatex was studied using various techniques comprising atomic force microscopy (AFM) and laser Doppler velocimetry (LDV). Initially, the basic physicochemical properties of IgaR molecules and the particles, inter alia their electrophoretic mobilities, the zeta potentials and hydrodynamic diameters, were determined under different ionic strengths and pHs. Applying AFM, single immunoglobulin molecules adsorbed on mica were also imaged, which allowed to determine their size. The adsorption of the IgaR molecules on the particles leading to changes in their electrophoretic mobility was monitored in situ using the LDV method. The obtained results were interpreted applying a general electrokinetic model which yielded quantitative information about the molecule coverage on the particles. The obtained immunolatex was thoroughly characterized with respect to its acid-base properties and its stability upon storage. Notably, the developed procedure demonstrated better efficiency compared to commercially applied methods, characterized by a higher immunoglobulin consumption.

Physicochemical Nature of SARS-CoV-2 Spike Protein Binding to Human Vimentin.

Vimentin, a protein that builds part of the cytoskeleton and is involved in many aspects of cellular function, was recently identified as a cell surface attachment site for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The present study investigated the physicochemical nature of the binding between the SARS-CoV-2 S1 glycoprotein receptor binding domain (S1 RBD) and human vimentin using atomic force microscopy and a quartz crystal microbalance. The molecular interactions of S1 RBD and vimentin proteins were quantified using vimentin monolayers attached to the cleaved mica or a gold microbalance sensor as well as in its native extracellular form present on the live cell surface. The presence of specific interactions between vimentin and S1 RBD was also confirmed using in silico studies. This work provides new evidence that cell-surface vimentin (CSV) functions as a site for SARS-CoV-2 virus attachment and is involved in the pathogenesis of Covid-19, providing a potential target for therapeutic countermeasures.

Mimicking Pseudo-Virion Interactions with Abiotic Surfaces: Deposition of Polymer Nanoparticles with Albumin Corona.

Adsorption of human serum albumin (HSA) molecules on negatively charged polystyrene microparticles was studied using the dynamic light scattering, the electrophoretic and the solution depletion methods involving atomic force microscopy. Initially, the physicochemical characteristics of the albumin comprising the hydrodynamic diameter, the zeta potential and the isoelectric point were determined as a function of pH. Analogous characteristics of the polymer particles were acquired, including their size and zeta potential. The formation of albumin corona on the particles was investigated in situ by electrophoretic mobility measurements. The size, stability and electrokinetic properties of the particles with the corona were also determined. The particle diameter was equal to 125 nm, which coincides with the size of the SARS-CoV-2 virion. The isoelectric point of the particles appeared at a pH of 5. The deposition kinetics of the particles was determined by atomic force microscopy (AFM) under diffusion and by quartz microbalance (QCM) under flow conditions. It was shown that the deposition rate at a gold sensor abruptly vanished with pH following the decrease in the zeta potential of the particles. It is postulated that the acquired results can be used as useful reference systems mimicking virus adsorption on abiotic surfaces.

SARS-CoV-2 Spike Protein (RBD) Subunit Adsorption at Abiotic Surfaces and Corona Formation at Polymer Particles.

The adsorption kinetics of the SARS-CoV-2 spike protein subunit with the receptor binding domain at abiotic surfaces was investigated. A combination of sensitive methods was used such as atomic force microscopy yielding a molecular resolution, a quartz microbalance, and optical waveguide lightmode spectroscopy. The two latter methods yielded in situ information about the protein adsorption kinetics under flow conditions. It was established that at pH 3.5-4 the protein adsorbed on mica and silica surfaces in the form of compact quasi-spherical aggregates with an average size of 14 nm. The maximum coverage of the layers was equal to 3 and 1 mg m-2 at pH 4 and 7.4, respectively. The experimental data were successfully interpreted in terms of theoretical results derived from modeling. The experiments performed for flat substrates were complemented by investigations of the protein corona formation at polymer particles carried out using in situ laser Doppler velocimetry technique. In this way, the zeta potential of the protein layers was acquired as a function of the coverage. Applying the electrokinetic model, these primary data were converted to the dependence of the subunit zeta potential on pH. It was shown that a complete acid-base characteristic of the layer can be acquired only using nanomolar quantities of the protein.

QCM-D Investigations of Anisotropic Particle Deposition Kinetics: Evidences of the Hydrodynamic Slip Mechanisms.

Deposition kinetics of positively charged polymer microparticles, characterized by prolate spheroid shape, at silica and gold sensors was investigated using the quartz microbalance (QCM) technique. Reference measurements were also performed for positively charged polymer particles of spherical shape and the same mass as the spheroids. Primarily, the frequency and bandwidth shifts for various overtones were measured as a function of time. It is shown that the ratio of these signals is close to unity for all overtones. These results were converted to the dependence of the frequency shift on the particle coverage, directly determined by atomic force microscopy and theoretically interpreted in terms of the hydrodynamic model. A quantitative agreement with experiments was attained considering particle slip relative to the ambient oscillating flow. In contrast, the theoretical results pertinent to the rigid contact model proved inadequate. The particle deposition kinetics derived from the QCM method was compared with theoretical modeling performed according to the random sequential adsorption approach. This allowed to assess the feasibility of the QCM technique to furnish proper deposition kinetics for anisotropic particles. It is argued that the hydrodynamic slip effect should be considered in the interpretation of QCM kinetic results acquired for bioparticles, especially viruses.

Human Vimentin Layers on Solid Substrates: Adsorption Kinetics and Corona Formation Investigations.

Adsorption kinetics of human vimentin on negatively charged substrates (mica, silica, and polymer particles) was analyzed using atomic force microscopy (AFM), quartz microbalance (QCM), and the laser doppler velocimetry (LDV) method. AFM studies realized under diffusion conditions proved that the adsorbed protein layer mainly consisted of aggregates in the form of compact tetramers and hexamers of a size equal to 11-12 nm. These results were consistent with vimentin adsorption kinetics under flow conditions investigated by QCM. It was established that vimentin aggregates efficiently adsorbed on the negatively charged silica sensor at pH 3.5 and 7.4, forming compact layers with the coverage reaching 3.5 mg m-2. Additionally, the formation of the vimentin corona at polymer particles was examined using the LDV method and interpreted in terms of the electrokinetic model. This allowed us to determine the zeta potential of the corona as a function of pH and the electrokinetic charge of aggregates, which was equal to -0.7 e nm-2 at pH 7.4 in a 10 mM NaCl solution. The anomalous adsorption of aggregates exhibiting an average negative charge on the negatively charged substrates was interpreted as a result of a heterogeneous charge distribution. These investigations confirmed that it is feasible to deposit stable vimentin layers both at planar substrates and at carrier particles with well-controlled coverage and zeta potential. They can be used for investigations of vimentin interactions with various ligands including receptors of the innate immune system, immunoglobulins, bacterial virulence factors, and spike proteins of viruses.

Chitosan characteristics in electrolyte solutions: Combined molecular dynamics modeling and slender body hydrodynamics.

Molecular dynamics modeling was applied to predict chitosan molecule conformations, the contour length, the gyration radius, the effective cross-section and the density in electrolyte solutions. Using various experimental techniques the diffusion coefficient, the hydrodynamic diameter and the electrophoretic mobility of molecules were determined. This allowed to calculate the zeta potential, the electrokinetic charge and the effective ionization degree of the chitosan molecule as a function of pH and the temperature. The chitosan solution density and zero shear dynamic viscosity were also measured, which enabled to determine the intrinsic viscosity increment. The experimental results were quantitatively interpreted in terms of the slender body hydrodynamics exploiting molecule characteristics derived from the modeling. It is also confirmed that this approach can be successfully used for a proper interpretation of previous literature data obtained under various physicochemical conditions.

Nanoparticle and bioparticle deposition kinetics.

Mechanisms and kinetic of particle deposition at solid surfaces leading to the formation of self-assembled layers of controlled structure and density were reviewed. In the first part theoretical aspects were briefly discussed, comprising limiting analytical solutions for the linear transport under flow and diffusion. Methods of the deposition kinetics analysis for non-linear regimes affected by surface blocking were also considered. Characteristic monolayer formation times under diffusion and flow for the nanoparticle size range were calculated. In the second part illustrative experimental results obtained for micro- and nanoparticles were discussed. Deposition at planar substrates was analyzed with emphasis focused on the stability of layers and the release kinetics of silver particles. Applicability of the quartz microbalance measurements (QCM) for quantitative studies of nanoparticle deposition kinetic was also discussed. Except for noble metal and polymer particles, representative results for virus deposition at abiotic surfaces were analyzed. Final part of the review was devoted to nanoparticle corona formation at polymer carrier particles investigated by combination of the concentration depletion, AFM, SEM and the in situ electrokinetic method. It is argued that the results obtained for colloid particles can be used as reliable reference systems for interpretation of protein and other bioparticle deposition, confirming the thesis that simple is universal.

Poly-L-Arginine Molecule Properties in Simple Electrolytes: Molecular Dynamic Modeling and Experiments.

Physicochemical properties of poly-L-arginine (P-Arg) molecules in NaCl solutions were determined by molecular dynamics (MD) modeling and various experimental techniques. Primarily, the molecule conformations, the monomer length and the chain diameter were theoretically calculated. These results were used to interpret experimental data, which comprised the molecule secondary structure, the diffusion coefficient, the hydrodynamic diameter and the electrophoretic mobility determined at various ionic strengths and pHs. Using these data, the electrokinetic charge and the effective ionization degree of P-Arg molecules were determined. In addition, the dynamic viscosity measurements for dilute P-Arg solutions enabledto determine the molecule intrinsic viscosity, which was equal to 500 and 90 for ionic strength of 10-5 and 0.15 M, respectively. This confirmed that P-Arg molecules assumed extended conformations and approached the slender body limit at the low range of ionic strength. The experimental data were also used to determine the molecule length and the chain diameter, which agreed with theoretical predictions. Exploiting these results, a robust method for determining the molar mass of P-Arg samples, the hydrodynamic diameter, the radius of gyration and the sedimentation coefficient was proposed.

Kinetics of Immunolatex Deposition at Abiotic Surfaces under Flow Conditions: Towards Quantitative Agglutination Assays.

Physicochemical properties of immunolatex, prepared by incubation of negatively charged polystyrene microparticles with polyclonal rabbit IgGs, were determined by a variety of experimental techniques. These comprised dynamic light scattering (DLS), laser Doppler velocimetry (LDV) and atomic force microscopy (AFM). The particle diffusion coefficient, the hydrodynamic diameter, the electrophoretic mobility, the zeta potential and the suspension stability were determined as a function of pH for different ionic strengths. The deposition of the immunolatex on bare and polyallylamine (PAH) functionalized mica was investigated using the microfluidic oblique impinging-jet cell, with an in situ, real-time image analysis module. The particle deposition kinetics was acquired by a direct particle enumeration procedure. The measurements enabled us to determine the range of pH where the specific deposition of the immunolatex on these substrates was absent. We argue that the obtained results have practical significance for conducting efficient flow immunoassays governed by specific antigen/antibody interactions.

Deposition of Polymer Particles with Fibrinogen Corona at Abiotic Surfaces under Flow Conditions.

The deposition kinetics of polymer particles with fibrinogen molecule coronas at bare and poly-L-lysine (PLL) modified mica was studied using the microfluid impinging-jet cell. Basic physicochemical characteristics of fibrinogen and the particles were acquired using dynamic light scattering and the electrophoretic mobility methods, whereas the zeta potential of the substrates was determined using streaming potential measurements. Subsequently, an efficient method for the preparation of the particles with coronas, characterized by a controlled fibrinogen coverage, was developed. This enabled us to carry out measurements, which confirmed that the deposition kinetics of the particles at mica vanished at pH above 5. In contrast, the particle deposition of PLL modified mica was at maximum for pH above 5. It was shown that the deposition kinetics could be adequately analyzed in terms of the mean-field approach, analogously to the ordinary colloid particle behavior. This contrasts the fibrinogen molecule behavior, which efficiently adsorbs at negatively charged substrates for the entire range pHs up to 9.7. These results have practical significance for conducting label-free immunoassays governed by the specific antigen/antibody interactions.

Mechanism of Myoglobin Molecule Adsorption on Silica: QCM, OWLS and AFM Investigations.

Adsorption kinetics of myoglobin on silica was investigated using the quartz crystal microbalance (QCM) and the optical waveguide light-mode spectroscopy (OWLS). Measurements were carried out for the NaCl concentration of 0.01 M and 0.15 M. A quantitative analysis of the kinetic adsorption and desorption runs acquired from QCM allowed to determine the maximum coverage of irreversibly bound myoglobin molecules. At a pH of 3.5-4 this was equal to 0.60 mg m-2 and 1.3 mg m-2 for a NaCl concentration of 0.01 M and 0.15 M, respectively, which agrees with the OWLS measurements. The latter value corresponds to the closely packed monolayer of molecules predicted from the random sequential adsorption approach. The fraction of reversibly bound protein molecules and their biding energy were also determined. It is observed that at larger pHs, the myoglobin adsorption kinetics was much slower. This behavior was attributed to the vanishing net charge that decreased the binding energy of molecules with the substrate. These results can be exploited to develop procedures for preparing myoglobin layers at silica substrates of well-controlled coverage useful for biosensing purposes.

SARS-CoV-2 virion physicochemical characteristics pertinent to abiotic substrate attachment.

The structure, size, and main physicochemical characteristics of the SARS-CoV-2 virion with the spike transmembrane protein corona were discussed. Using these data, diffusion coefficients of the virion in aqueous media and in air were calculated. The structure and dimensions of the spike protein derived from molecular dynamic modeling and thorough cryo-electron microscopy measurements were also analyzed. The charge distribution over the molecule was calculated and shown to be largely heterogeneous. Although the stalk part is negatively charged, the top part of the spike molecule, especially the receptor binding domain, remains positively charged for a broad range of pH. It is underlined that such a charge distribution promotes the spike corona stability and enhances the virion attachment to receptors and surfaces, mostly negatively charged. The review is completed by the analysis of experimental data pertinent to the spike protein adsorption at abiotic surfaces comprising nanoparticle carrier particles. It is argued that these theoretical and experimental data can be used for developing quantitative models of virus attachment to surfaces, facilitating adequate analysis of future experimental results.