AFM for Studying the Functional Activity of Enzymes Irina A. Ivanova, Anastasia A. Valueva, Maria O. Ershova, Tatiana O. Pleshakova Biomolecules, 2025 The conventional approach to investigating enzyme systems involves the simultaneous investigation of a large number of molecules and observing ensemble-averaged properties. However, modern science allows us to study the properties of single molecules and to obtain data on biochemical systems at a fundamentally new level, significantly expanding our understanding of the mechanisms of biochemical processes. Imaging of single biomolecules with high spatial and temporal resolution is among such modern research tools. To effectively image the individual steps or intermediates of biochemical reactions in single-molecule experiments, we need to develop a methodology for data acquisition and analysis. Its development will make it possible to solve the problem of separating the static and dynamic disorder present in the parameters identified by traditional proteomic methods. Such a methodology may be based on AFM imaging, the high-resolution microscopic visualization of enzymes. This review focuses on this direction of research, including the relevant methodological and practical solutions related to the potential of developing a single-molecule approach to the study of enzyme systems using AFM-based techniques. We focus on the results of enzyme reaction studies, as there are still few such studies, as opposed to the AFM studies of the mechanical properties of individual enzyme molecules.
Using the Radial Distribution Function to Analyze Atomic Force Microscopy Images of Colloidal Systems Sergey V. Kraevsky, Anastasia A. Valueva, Maria O. Ershova, Ivan D. Shumov, Irina A. Ivanova, et al. International Journal of Molecular Sciences, 2025 Biomacromolecules generally exist and function in aqueous media. Is it possible to estimate the state and properties of molecules in an initial three-dimensional colloidal solution based on the structure properties of biomolecules adsorbed on the two-dimensional surface? Using atomic force microscopy to study nanosized objects requires their immobilization on a surface. Particles undergoing Brownian motion in a solution significantly reduce their velocity near the surface and become completely immobilized upon drying. Using radial distribution function (RDF) methods, it is possible to obtain information about the presence of short-range or long-range order in the arrangement of immobilized colloidal particles. In this work, RDF is applied to immobilized gold nanoparticles (AuNPs) and horseradish peroxidase molecules on mica. It is shown that AuNPs maintain mobility on the mica surface when water is present. Upon immobilization, AuNPs organize into an amorphous structure exhibiting short-range order. Protein molecules are immobilized randomly, and their surface density is well described by the Poisson distribution.
Atomic Force Microscopy Study of the Long-Term Effect of the Glycerol Flow, Stopped in a Coiled Heat Exchanger, on Horseradish Peroxidase Yuri D. Ivanov, Ivan D. Shumov, Andrey F. Kozlov, Anastasia A. Valueva, Maria O. Ershova, et al. Micromachines, 2024 Glycerol is employed as a functional component of heat-transfer fluids, which are of use in both bioreactors and various biosensor devices. At the same time, flowing glycerol was reported to cause considerable triboelectric effects. Herein, by using atomic force microscopy (AFM), we have revealed the long-term effect of glycerol flow, stopped in a ground-shielded coiled heat exchanger, on horseradish peroxidase (HRP) adsorption on mica. Namely, the solution of HRP was incubated in the vicinity of the side of the cylindrical coil with stopped glycerol flow, and then HRP was adsorbed from this solution onto a mica substrate. This incubation has been found to markedly increase the content of aggregated enzyme on mica—as compared with the control enzyme sample. We explain the phenomenon observed by the influence of triboelectrically induced electromagnetic fields of non-trivial topology. The results reported should be further considered in the development of flow-based heat exchangers of biosensors and bioreactors intended for operation with enzymes.
AFM-FISHING TECHNOLOGY FOR PROTEIN DETECTION IN SOLUTIONS T.O. Pleshakova, M.O. Ershova, A.A. Valueva, I.A. Ivanova, Yu.D. Ivanov, et al. Biomeditsinskaya Khimiya, 2024 The review considers the possibility of using atomic force microscopy (AFM) as a basic method for protein detection in solutions with low protein concentrations. The demand for new bioanalytical approaches is determined by the problem of insufficient sensitivity of systems used in routine practice for protein detection. Special attention is paid to demonstration of the use in bioanalysis of a combination of AFM and fishing methods as an approach of concentrating biomolecules from a large volume of the analyzed solution on a small surface area.
MS Identification of Blood Plasma Proteins Concentrated on a Photocrosslinker-Modified Surface Arina I. Gordeeva, Anastasia A. Valueva, Elizaveta E. Rybakova, Maria O. Ershova, Ivan D. Shumov, et al. International Journal of Molecular Sciences, 2024 This work demonstrates the use of a modified mica to concentrate proteins, which is required for proteomic profiling of blood plasma by mass spectrometry (MS). The surface of mica substrates, which are routinely used in atomic force microscopy (AFM), was modified with a photocrosslinker to allow “irreversible” binding of proteins via covalent bond formation. This modified substrate was called the AFM chip. This study aimed to determine the role of the surface and crosslinker in the efficient concentration of various types of proteins in plasma over a wide concentration range. The substrate surface was modified with a 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) photocrosslinker, activated by UV irradiation. AFM chips were incubated with plasma samples from a healthy volunteer at various dilution ratios (102X, 104X, and 106X). Control experiments were performed without UV irradiation to evaluate the contribution of physical protein adsorption to the concentration efficiency. AFM imaging confirmed the presence of protein layers on the chip surface after incubation with the samples. MS analysis of different samples indicated that the proteomic profile of the AFM-visualized layers contained common and unique proteins. In the working series of experiments, 228 proteins were identified on the chip surface for all samples, and 21 proteins were not identified in the control series. In the control series, a total of 220 proteins were identified on the chip surface, seven of which were not found in the working series. In plasma samples at various dilution ratios, a total of 146 proteins were identified without the concentration step, while 17 proteins were not detected in the series using AFM chips. The introduction of a concentration step using AFM chips allowed us to identify more proteins than in plasma samples without this step. We found that AFM chips with a modified surface facilitate the efficient concentration of proteins owing to the adsorption factor and the formation of covalent bonds between the proteins and the chip surface. The results of our study can be applied in the development of highly sensitive analytical systems for determining the complete composition of the plasma proteome.
Selection of Aptamers for Use as Molecular Probes in AFM Detection of Proteins Maria O. Ershova, Amir Taldaev, Petr V. Konarev, Georgy S. Peters, Anastasia A. Valueva, et al. Biomolecules, 2023 Currently, there is great interest in the development of highly sensitive bioanalytical systems for diagnosing diseases at an early stage, when pathological biomarkers are present in biological fluids at low concentrations and there are no clinical manifestations. A promising direction is the use of molecular detectors―highly sensitive devices that detect signals from single biomacromolecules. A typical detector in this class is the atomic force microscope (AFM). The high sensitivity of an AFM-based bioanalysis system is determined by the size of the sensing element of an atomic force microscope―the cantilever―the radius of the curvature of which is comparable to that of a biomolecule. Biospecific molecular probe–target interactions are used to ensure detection system specificity. Antibodies, aptamers, synthetic antibodies, and peptides can be used as molecular probes. This study has demonstrated the possibility of using aptamers as molecular probes for AFM-based detection of the ovarian cancer biomarker CA125. Antigen detection in a nanomolar solution was carried out using AFM chips with immobilized aptamers, commercially available or synthesized based on sequences from open sources. Both aptamer types can be used for antigen detection, but the availability of sequence information enables additional modeling of the aptamer structure with allowance for modifications necessary for immobilization of the aptamer on an AFM chip surface. Information on the structure and oligomeric composition of aptamers in the solution was acquired by combining small-angle X-ray scattering and molecular modeling. Modeling enabled pre-selection, before the experimental stage, of aptamers for use as surface-immobilized molecular probes.
Glycerol Flow through a Shielded Coil Induces Aggregation and Activity Enhancement of Horseradish Peroxidase Yuri D. Ivanov, Ivan D. Shumov, Andrey F. Kozlov, Maria O. Ershova, Anastasia A. Valueva, et al. Applied Sciences Switzerland, 2023 Glycerol has found its applications as a heat-transfer fluid in heat exchangers, and as a component of functional liquids in biosensor analysis. Flowing non-aqueous fluids are known to be able to induce electromagnetic fields due to the triboelectric effect. These triboelectrically generated electromagnetic fields can affect biological macromolecules. Horseradish peroxidase (HRP) is widely employed as a convenient model object for studying how external electric, magnetic, and electromagnetic fields affect enzymes. Herein, we have studied whether the flow of glycerol in a ground-shielded cylindrical coil affects the HRP enzyme incubated at a 2 cm distance near the coil’s side. Atomic force microscopy (AFM) has been employed in order to study the effect of glycerol flow on HRP at the nanoscale. An increased aggregation of HRP on mica has been observed after the incubation of the enzyme near the coil. Moreover, the enzymatic activity of HRP has also been affected. The results reported that their application can be found in biotechnology, food technology and life sciences applications, considering the development of triboelectric generators, enzyme-based biosensors and bioreactors with surface-immobilized enzymes. Our work can also be of interest for scientists studying triboelectric phenomena, representing one more step toward understanding the mechanism of the indirect action of the flow of a dielectric liquid on biological macromolecules.
Stopped Flow of Glycerol Induces the Enhancement of Adsorption and Aggregation of HRP on Mica Yuri D. Ivanov, Ivan D. Shumov, Andrey F. Kozlov, Maria O. Ershova, Anastasia A. Valueva, et al. Micromachines, 2023 Glycerol is a usable component of heat-transfer fluids, and is thus suitable for the use in microchannel-based heat exchangers in biosensors and microelectronic devices. The flow of a fluid can lead to the generation of electromagnetic fields, which can affect enzymes. Herein, by means of atomic force microscopy (AFM) and spectrophotometry, a long-term effect of stopped flow of glycerol through a coiled heat exchanger on horseradish peroxidase (HRP) has been revealed. Samples of buffered HRP solution were incubated near either the inlet or the outlet sections of the heat exchanger after stopping the flow. It has been found that both the enzyme aggregation state and the number of mica-adsorbed HRP particles increase after such an incubation for 40 min. Moreover, the enzymatic activity of the enzyme incubated near the inlet section has been found to increase in comparison with that of the control sample, while the activity of the enzyme incubated near the outlet section remained unaffected. Our results can find application in the development of biosensors and bioreactors, in which flow-based heat exchangers are employed.
