Maria Ershova
@en.ibmc.msk.ru
Institute of Biomedical Chemistry
RESEARCH, TEACHING, or OTHER INTERESTS
Biochemistry, Structural Biology, Immunology, Biochemistry
Scopus Publications
Scopus Publications
Yuri D. Ivanov, Ivan D. Shumov, Andrey F. Kozlov, Anastasia A. Valueva, Maria O. Ershova, Irina A. Ivanova, Alexander N. Ableev, Vadim Y. Tatur, Andrei A. Lukyanitsa, Nina D. Ivanova,et al.
MDPI AG
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.
Arina I. Gordeeva, Anastasia A. Valueva, Elizaveta E. Rybakova, Maria O. Ershova, Ivan D. Shumov, Andrey F. Kozlov, Vadim S. Ziborov, Anna S. Kozlova, Victor G. Zgoda, Yuri D. Ivanov,et al.
MDPI AG
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.
Maria O. Ershova, Amir Taldaev, Petr V. Konarev, Georgy S. Peters, Anastasia A. Valueva, Irina A. Ivanova, Sergey V. Kraevsky, Andrey F. Kozlov, Vadim S. Ziborov, Yuri D. Ivanov,et al.
MDPI AG
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.
Yuri D. Ivanov, Ivan D. Shumov, Andrey F. Kozlov, Maria O. Ershova, Anastasia A. Valueva, Irina A. Ivanova, Vadim Y. Tatur, Andrei A. Lukyanitsa, Nina D. Ivanova, and Vadim S. Ziborov
MDPI AG
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.
Yuri D. Ivanov, Ivan D. Shumov, Andrey F. Kozlov, Maria O. Ershova, Anastasia A. Valueva, Irina A. Ivanova, Vadim Y. Tatur, Andrei A. Lukyanitsa, Nina D. Ivanova, and Vadim S. Ziborov
MDPI AG
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.
Arina I. Gordeeva, Anastasia A. Valueva, Maria O. Ershova, Elizaveta E. Rybakova, Ivan D. Shumov, Andrey F. Kozlov, Vadim S. Ziborov, Maria G. Zavialova, Victor G. Zgoda, Yuri D. Ivanov,et al.
MDPI AG
Mass spectrometry (MS) is one of the main techniques for protein identification. Herein, MS has been employed for the identification of bovine serum albumin (BSA), which was covalently immobilized on the surface of a mica chip intended for investigation by atomic force microscopy (AFM). For the immobilization, two different types of crosslinkers have been used: 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) and dithiobis(succinimidyl propionate) (DSP). According to the data obtained by using an AFM-based molecular detector, the SuccBB crosslinker was more efficient in BSA immobilization than the DSP. The type of crosslinker used for protein capturing has been found to affect the results of MS identification. The results obtained herein can be applied in the development of novel systems intended for the highly sensitive analysis of proteins with molecular detectors.
Yuri D. Ivanov, Vadim Y. Tatur, Ivan D. Shumov, Andrey F. Kozlov, Anastasia A. Valueva, Irina A. Ivanova, Maria O. Ershova, Nina D. Ivanova, Igor N. Stepanov, Andrei A. Lukyanitsa,et al.
MDPI AG
In this research, the influence of a dodecahedron-shaped structure on the adsorption behavior of a horseradish peroxidase (HRP) enzyme glycoprotein onto mica substrates was studied. In the experiments, samples of an aqueous HRP solution were incubated at various distances (0.03 m, 2 m, 5 m, and control at 20 m) from the dodecahedron surface. After the incubation, the direct adsorption of HRP onto mica substrates immersed in the solutions was performed, and the mica-adsorbed HRP particles were visualized via atomic force microscopy (AFM). The effect of the increased HRP aggregation was only observed after the incubation of the enzyme solution at the 2 m distance from the dodecahedron. In addition, with respect to the control sample, spectrophotometric measurements revealed no change in the HRP enzymatic activity after the incubation at any of the distances studied. The results reported herein can be of use in the modeling of the possible influences of various spatial structures on biological objects in the development of biosensors and other electronic equipment.
Yuri D. Ivanov, Vadim Y. Tatur, Ivan D. Shumov, Andrey F. Kozlov, Anastasia A. Valueva, Irina A. Ivanova, Maria O. Ershova, Nina D. Ivanova, Igor N. Stepanov, Andrei A. Lukyanitsa,et al.
MDPI AG
The influence of an external constant strong electric field, formed using a pyramidal structure under a high electric potential, on an enzyme located near its apex, is studied. Horseradish peroxidase (HRP) is used as a model. In our experiments, a 27 kV direct current (DC) voltage was applied to two electrodes with a conducting pyramidal structure attached to one of them. The enzyme particles were visualized by atomic force microscopy (AFM) after the adsorption of the enzyme from its 0.1 µM solution onto mica AFM substrates. It is demonstrated that after the 40 min exposure to the electric field, the enzyme forms extended structures on mica, while in control experiments compact HRP particles are observed. After the exposure to the electric field, the majority of mica-adsorbed HRP particles had a height of 1.2 nm (as opposed to 1.0 nm in the case of control experiments), and the contribution of higher (>2.0 nm) particles was also considerable. This indicates the formation of high-order HRP aggregates under the influence of an applied electric field. At that, the enzymatic activity of HRP against its substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) remains unaffected. These results are important for studying macroscopic effects of strong electromagnetic fields on enzymes, as well as for the development of cellular structure models.
Yuri D. Ivanov, Ivan D. Shumov, Vadim Y. Tatur, Anastasia A. Valueva, Andrey F. Kozlov, Irina A. Ivanova, Maria O. Ershova, Nina D. Ivanova, Igor N. Stepanov, Andrei A. Lukyanitsa,et al.
MDPI AG
The present study is aimed at the revelation of subtle effects of steam flow through a conical coil heat exchanger on an enzyme, incubated near the heat exchanger, at the nanoscale. For this purpose, atomic force microscopy (AFM) has been employed. In our experiments, horseradish peroxidase (HRP) was used as a model enzyme. HRP is extensively employed as a model in food science in order to determine the influence of electromagnetic fields on enzymes. Adsorption properties of HRP on mica have been studied by AFM at the level of individual enzyme macromolecules, while the enzymatic activity of HRP has been studied by spectrophotometry. The solution of HRP was incubated either near the top or at the side of the conically wound aluminium pipe, through which steam flow passed. Our AFM data indicated an increase in the enzyme aggregation on mica after its incubation at either of the two points near the heat exchanger. At the same time, in the spectrophotometry experiments, a slight change in the shape of the curves, reflecting the HRP-catalyzed kinetics of ABTS oxidation by hydrogen peroxide, has also been observed after the incubation of the enzyme solution near the heat exchanger. These effects on the enzyme adsorption and kinetics can be explained by alterations in the enzyme hydration caused by the influence of the electromagnetic field, induced triboelectrically by the flow of steam through the heat exchanger. Our findings should thus be considered in the development of equipment involving conical heat exchangers, intended for either research or industrial use (including miniaturized bioreactors and biosensors). The increased aggregation of the HRP enzyme, observed after its incubation near the heat exchanger, should also be taken into account in analysis of possible adverse effects from steam-heated industrial equipment on the human body.
Yuri D. Ivanov, Vadim Y. Tatur, Ivan D. Shumov, Andrey F. Kozlov, Anastasia A. Valueva, Irina A. Ivanova, Maria O. Ershova, Nina D. Ivanova, Igor N. Stepanov, Andrei A. Lukyanitsa,et al.
MDPI AG
The effect of a dielectric conical structure on the adsorption properties of an enzyme on mica was studied by atomic force microscopy (AFM) with the example of horseradish peroxidase (HRP). The cone used was a cellulose cone with a 60° apex angle. Namely, AFM allowed us to reveal an increase in the enzyme’s aggregation during its adsorption onto mica from the solution incubated near the cone apex for 40 min—as compared with the control enzyme samples incubated far away from the cone. In contrast, no change in the HRP adsorption properties was observed after shorter (10 min) incubation of the sample near the cone. The enzymatic activity of HRP was found to be the same for all the enzyme samples studied. Our findings should be considered upon designing biosensors (in particular, those intended for highly sensitive diagnostic applications) and bioreactors containing conical structural elements. Furthermore, since HRP is widely employed as a model enzyme in studies of external impacts on enzymes determining food quality, our data can be of use in the development of food-processing methods based on the use of electromagnetic radiation (microwave treatment, radiofrequency heating, etc.).
Yuri D. Ivanov, Vadim Y. Tatur, Ivan D. Shumov, Andrey F. Kozlov, Anastasia A. Valueva, Irina A. Ivanova, Maria O. Ershova, Nina D. Ivanova, Igor N. Stepanov, Andrei A. Lukyanitsa,et al.
MDPI AG
Our study reported herein aims to determine whether an electromagnetic field, induced triboelectrically by a metallic cone, rotating at a frequency of 167 Hz, has an effect on the properties of the horseradish peroxidase (HRP) enzyme. Atomic force microscopy (AFM) was employed to detect even the most subtle effects on single enzyme molecules. In parallel, a macroscopic method (spectrophotometry) was used to reveal whether the enzymatic activity of HRP in solution was affected. An aqueous solution of the enzyme was incubated at a distance of 2 cm from the rotating cone. The experiments were performed at various incubation times. The control experiments were performed with a non-rotating cone. The incubation of the HRP solution was found to cause the disaggregation of the enzyme. At longer incubation times, this disaggregation was found to be accompanied by the formation of higher-order aggregates; however, no change in the HRP enzymatic activity was observed. The results of our experiments could be of interest in the development of enzyme-based biosensors with rotating elements such as stirrers. Additionally, the results obtained herein are important for the correct interpretation of data obtained with such biosensors.
Yuri D. Ivanov, Vadim Yu. Tatur, Ivan D. Shumov, Andrey F. Kozlov, Anastasia A. Valueva, Irina A. Ivanova, Maria O. Ershova, Nina D. Ivanova, Igor N. Stepanov, Andrei A. Lukyanitsa,et al.
MDPI AG
The effect of a high-voltage discharge in a helicoidal structure on the adsorption properties of an enzyme on mica has been studied with the example of horseradish peroxidase (HRP). The discharge was generated at the expense of a sparkover in a 3 mm gap between two electrodes, to which a 10 kV, 50 Hz AC voltage was applied. The electrodes were connected to a twisted pair, which was wound onto a cone, forming the helicoidal structure. The incubation of the enzyme solution near the top of the helicoidal structure has been found to cause an increase in the degree of aggregation of HRP adsorbed on mica in comparison with the control HRP sample. The results obtained should be taken into account in studies of enzymes using biosensors with helicoidal structures as heating elements, as well as in refining models describing effects of low-frequency alternating current, flowing through helicoidal structures, on proteins and biological objects.
Irina A. Ivanova, Maria O. Ershova, Ivan D. Shumov, Anastasia A. Valueva, Yuri D. Ivanov, and Tatyana O. Pleshakova
MDPI AG
This paper presents an investigation of the temperature dependence of the oligomeric state of the horseradish peroxidase (HRP) enzyme on the temperature of its solution, and on the solution storage time, at the single-molecule level. Atomic force microscopy has been employed to determine how the temperature and the storage time of the HRP solution influence its aggregation upon direct adsorption of the enzyme from the solution onto bare mica substrates. In parallel, spectrophotometric measurements have been performed in order to estimate whether the HRP enzymatic activity changes over time upon the storage of the enzyme solution. The temperature dependence of the HRP oligomeric state has been studied within a broad (15–40 °C) temperature range. It has been demonstrated that the storage of the HRP solution for 14 days does not have any considerable effect on the oligomeric state of the enzyme, neither does it affect its activity. At longer storage times, AFM has allowed us to reveal a tendency of HRP to oligomerization during the storage of its buffered solution, while the enzymatic activity remains virtually unchanged even after a 1-month-long storage. By AFM, it has been revealed that after the incubation of a mica substrate in the HRP solution at various temperatures, the content of the mica-adsorbed oligomers increases insignificantly owing to a high-temperature stability of the enzyme.
Yuri D. Ivanov, Vadim Yu. Tatur, Tatyana O. Pleshakova, Ivan D. Shumov, Andrey F. Kozlov, Anastasia A. Valueva, Irina A. Ivanova, Maria O. Ershova, Nina D. Ivanova, Igor N. Stepanov,et al.
MDPI AG
The incubation of a solution of horseradish peroxidase (HRP) enzyme either below the apex or near the base of an inversely oriented square pyramid (inverted square pyramid; ISP) has been found to influence the enzyme’s aggregation and adsorption properties. The HRP enzyme is used herein as a model object due to its importance in analytical chemistry applications. Atomic force microscopy (AFM) is employed to investigate the HRP’s adsorption on mica substrates at the single-molecule level. Conventional spectrophotometry is used in parallel as a reference method for the determination of the HRP’s enzymatic activity. Using AFM, we reveal a significant change in the adsorption properties of HRP on mica substrates after the incubation of the HRP solutions either above the base or below the apex of the ISP in comparison with the control HRP solution. The same situation is observed after the incubation of the enzyme solution above the center of the ISP’s base. Here, the enzymatic activity of HRP remained unaffected in both cases. Since pyramidal structures of positive and inverted orientation are employed in biosensor devices, it is important to take into account the results obtained herein in the development of highly sensitive biosensor systems, in which pyramidal structures are employed as sensor (such as AFM probes) or construction elements.
Vadim S. Ziborov, Tatyana O. Pleshakova, Ivan D. Shumov, Andrey F. Kozlov, Anastasia A. Valueva, Irina A. Ivanova, Maria O. Ershova, Dmitry I. Larionov, Alexey N. Evdokimov, Vadim Yu. Tatur,et al.
MDPI AG
Our present study concerns the influence of the picosecond rise-time-pulsed electromagnetic field, and the impact of nanosecond pulsed pressure on the aggregation state of horseradish peroxidase (HRP) as a model enzyme. The influence of a 640 kV/m pulsed electromagnetic field with a pulse rise-time of ~200 ps on the activity and aggregation state of an enzyme is studied by the single-molecule atomic force microscopy (AFM) method. The influence of such a field is shown to lead to aggregation of the protein and to a decrease in its enzymatic activity. Moreover, the effect of a shock wave with a pressure front rise-time of 80 ns on the increase in the HRP aggregation is demonstrated. The results obtained herein can be of use in modeling the impact of electromagnetic and pressure pulses on enzymes and on whole living organisms. Our results are also important for taking into account the effect of pulsed fields on the body in the development of drugs, therapeutic procedures, and novel highly sensitive medical diagnosticums.
Yuri D. Ivanov, Tatyana O. Pleshakova, Ivan D. Shumov, Andrey F. Kozlov, Irina A. Ivanova, Anastasia A. Valueva, Maria O. Ershova, Vadim Yu. Tatur, Igor N. Stepanov, Victor V. Repnikov,et al.
Springer Science and Business Media LLC
AbstractIn our present paper, the influence of a pyramidal structure on physicochemical properties of a protein in buffer solution has been studied. The pyramidal structure employed herein was similar to those produced industrially for anechoic chambers. Pyramidal structures are also used as elements of biosensors. Herein, horseradish peroxidase (HRP) enzyme was used as a model protein. HRP macromolecules were adsorbed from their solution onto an atomically smooth mica substrate, and then visualized by atomic force microscopy (AFM). In parallel, the enzymatic activity of HRP was estimated by conventional spectrophotometry. Additionally, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) has been employed in order to find out whether or not the protein secondary structure changes after the incubation of its solution either near the apex of a pyramid or in the center of its base. Using AFM, we have demonstrated that the incubation of the protein solution either in the vicinity of the pyramid’s apex or in the center of its base influences the physicochemical properties of the protein macromolecules. Namely, the incubation of the HRP solution in the vicinity of the top of the pyramidal structure has been shown to lead to an increase in the efficiency of the HRP adsorption onto mica. Moreover, after the incubation of the HRP solution either near the top of the pyramid or in the center of its base, the HRP macromolecules adsorb onto the mica surface predominantly in monomeric form. At that, the enzymatic activity of HRP does not change. The results of our present study are useful to be taken into account in the development of novel biosensor devices (including those for the diagnosis of cancer in humans), in which pyramidal structures are employed as sensor, noise suppression or construction elements.
Anna L. Kaysheva, Arina I. Isaeva, Tatyana O. Pleshakova, Ivan D. Shumov, Anastasia A. Valueva, Maria O. Ershova, Irina A. Ivanova, Vadim S. Ziborov, Ivan Y. Iourov, Svetlana G. Vorsanova,et al.
MDPI AG
MicroRNAs, which circulate in blood, are characterized by high diagnostic value; in biomedical research, they can be considered as candidate markers of various diseases. Mature microRNAs of glial cells and neurons can cross the blood–brain barrier and can be detected in the serum of patients with autism spectrum disorders (ASD) as components of macrovesicles, macromolecular protein and low-density lipoprotein particles. In our present study, we have proposed an approach, in which microRNAs in protein complexes can be concentrated on the surface of AFM chips with oligonucleotide molecular probes, specific against the target microRNAs. MicroRNAs, associated with the development of ASD in children, were selected as targets. The chips with immobilized molecular probes were incubated in serum samples of ASD patients and healthy volunteers. By atomic force microscopy (AFM), objects on the AFM chip surface have been revealed after incubation in the serum samples. The height of these objects amounted to 10 nm and 6 nm in the case of samples of ASD patients and healthy volunteers, respectively. MALDI-TOF-MS analysis of protein components on the chip surface allowed us to identify several cell proteins. These proteins are involved in the binding of nucleic acids (GBG10, RT24, RALYL), in the organization of proteasomes and nucleosomes (PSA4, NP1L4), and participate in the functioning of the channel of active potassium transport (KCNE5, KCNV2).
Yuri D. Ivanov, Vadim Yu. Tatur, Tatyana O. Pleshakova, Ivan D. Shumov, Andrey F. Kozlov, Anastasia A. Valueva, Irina A. Ivanova, Maria O. Ershova, Nina D. Ivanova, Victor V. Repnikov,et al.
MDPI AG
External electromagnetic fields are known to be able to concentrate inside the construction elements of biosensors and bioreactors owing to reflection from their surface. This can lead to changes in the structure of biopolymers (such as proteins), incubated inside these elements, thus influencing their functional properties. Our present study concerned the revelation of the effect of spherical elements, commonly employed in biosensors and bioreactors, on the physicochemical properties of proteins with the example of the horseradish peroxidase (HRP) enzyme. In our experiments, a solution of HRP was incubated within a 30 cm-diameter titanium half-sphere, which was used as a model construction element. Atomic force microscopy (AFM) was employed for the single-molecule visualization of the HRP macromolecules, adsorbed from the test solution onto mica substrates in order to find out whether the incubation of the test HRP solution within the half-sphere influenced the HRP aggregation state. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was employed in order to reveal whether the incubation of HRP solution within the half-sphere led to any changes in its secondary structure. In parallel, spectrophotometry-based estimation of the HRP enzymatic activity was performed in order to find out if the HRP active site was affected by the electromagnetic field under the conditions of our experiments. We revealed an increased aggregation of HRP after the incubation of its solution within the half-sphere in comparison with the control sample incubated far outside the half-sphere. ATR-FTIR allowed us to reveal alterations in HRP’s secondary structure. Such changes in the protein structure did not affect its active site, as was confirmed by spectrophotometry. The effect of spherical elements on a protein solution should be taken into account in the development of the optimized design of biosensors and bioreactors, intended for performing processes involving proteins in biomedicine and biotechnology, including highly sensitive biosensors intended for the diagnosis of socially significant diseases in humans (including oncology, cardiovascular diseases, etc.) at early stages.
Yuri D. Ivanov, Tatyana O. Pleshakova, Ivan D. Shumov, Andrey F. Kozlov, Irina A. Ivanova, Maria O. Ershova, Vadim Yu. Tatur, and Vadim S. Ziborov
MDPI AG
Flow-based coiled systems, through which a heat transfer fluid (such as glycerol) is pumped, are widely used for thermal stabilization of bioreactors and biosensor cuvettes and cells. Previously, using horseradish peroxidase (HRP) as a model protein, we have demonstrated that the incubation of a protein solution in a flow-based system over coiled pipe with flowing glycerol leads to a change in the adsorption properties of the protein macromolecules. Herein, we have studied the effect of the glycerol flow on the properties of HRP, the solution of which was placed differently: i.e., near either the inflow or the outflow linear sections of the pipe, while the coiled section of the pipe was shielded with a grounded metallic cover. Atomic force microscopy (AFM) has been employed in order to visualize the HRP protein macromolecules adsorbed from its solution onto the mica substrate surface. The quantity of adsorbed protein was estimated based on the AFM data. The enzymatic activity of HRP was estimated by spectrophotometry. We demonstrate that a change in the properties of HRP enzyme was observed after the incubation of its solution near the inflow/outflow linear sections of the pipe with flowing glycerol. Namely, after the incubation of HRP solution near the inflow section, a decrease in the protein adsorption onto mica was observed, but its enzymatic activity remained unchanged in comparison to the control sample. In another case, when the HRP solution was incubated near the outflow section, an increased protein adsorption was observed, while the enzyme exhibited considerably lower activity.
Yuri D. Ivanov, Tatyana O. Pleshakova, Ivan D. Shumov, Andrey F. Kozlov, Anastasia A. Valueva, Irina A. Ivanova, Maria O. Ershova, Dmitry I. Larionov, Victor V. Repnikov, Nina D. Ivanova,et al.
MDPI AG
Atomic force microscopy (AFM)-based fishing is a promising method for the detection of low-abundant proteins. This method is based on the capturing of the target proteins from the analyzed solution onto a solid substrate, with subsequent counting of the captured protein molecules on the substrate surface by AFM. Protein adsorption onto the substrate surface represents one of the key factors determining the capturing efficiency. Accordingly, studying the factors influencing the protein adsorbability onto the substrate surface represents an actual direction in biomedical research. Herein, the influence of water motion in a flow-based system on the protein adsorbability and on its enzymatic activity has been studied with an example of horseradish peroxidase (HRP) enzyme by AFM, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and conventional spectrophotometry. In the experiments, HRP solution was incubated in a setup modeling the flow section of a biosensor communication. The measuring cell with the protein solution was placed near a coiled silicone pipe, through which water was pumped. The adsorbability of the protein onto the surface of the mica substrate has been studied by AFM. It has been demonstrated that incubation of the HRP solution near the coiled silicone pipe with flowing water leads to an increase in its adsorbability onto mica. This is accompanied by a change in the enzyme’s secondary structure, as has been revealed by ATR-FTIR. At the same time, its enzymatic activity remains unchanged. The results reported herein can be useful in the development of models describing the influence of liquid flow on the properties of enzymes and other proteins. The latter is particularly important for the development of biosensors for biomedical applications—particularly for serological analysis, which is intended for the early diagnosis of various types of cancer and infectious diseases. Our results should also be taken into account in studies of the effects of protein aggregation on hemodynamics, which plays a key role in human body functioning.