The phenomenon of knotted electromagnetic field (KEMF) is now actively studied, as such fields are characterized by a nontrivial topology. The research in this field is mainly aimed at technical applications-for instance, the development of efficient communication systems. Until present, however, the influence of KEMF on biological objects (including enzyme systems) was not considered. Herein, we have studied the influence of KEMF on the aggregation and enzymatic activity of a protein with the example of horseradish peroxidase (HRP). The test HRP solution was irradiated in KEMF (the radiation power density was 10 −12 W/cm 2 at 2.3 GHz frequency) for 40 min. After the irradiation, the aggregation of HRP was examined by atomic force microscopy (AFM) at the single-molecule level. The enzymatic activity was monitored by conventional spectrophotometry. It has been demonstrated that an increased aggregation of HRP, adsorbed on the AFM substrate surface, was observed after irradiation of the protein sample in KEMF with low (10 −12 W/cm 2) radiation power density; at the same time, the enzymatic activity remained unchanged. the results obtained herein can be used in the development of models describing the interaction of enzymes with electromagnetic field. The obtained data can also be of importance considering possible pathological factors that can take place upon the influence of KEMF on biological objects-for instance, changes in hemodynamics due to increased protein aggregation are possible; the functionality of protein complexes can also be affected by aggregation of their protein subunits. These effects should also be taken into account in the development of novel highly sensitive systems for human serological diagnostics of breast cancer, prostate cancer, brain cancer and other oncological pathologies, and for diagnostics of diseases in animals, and crops. It is known that electromagnetic radiation of various intensity can have different influence on human body. Electromagnetic fields can have various topology, such as transverse and knotted one (knotted electromagnetic field, KEMF) 1,2. The simplest and most common electromagnetic waves are transverse ones, and, to date, their effects have been widely studied in various frequency and intensity ranges. As regards biomedical applications, microwave radiation is interesting in that, depending on its intensity, it is employed in biological research, and in both medical diagnostics and therapy. In this way, upon exposure of biological tissues to high-intensity radiation (~90 W/cm 2), their temperature increases to ~90 °С, and denaturation of biological objects is observed. It was shown that, under such conditions, partial loss of functional activity of proteins (for instance, peroxidase) is observed 3. At lower radiation intensity (10 μW/cm 2 4), both positive therapeutic effects (which usually take place owing to local heating 5) and negative effects are observed. Here, it should be noted that studies on the application of non-thermal effects of low-power microwave r...

The influence of liquid motion in flow-based systems on the aggregation state of an enzyme and on its enzymatic activity was studied, with horseradish peroxidase (HRP) as an example. Our experiments were carried out in a setup modeling the flow section of the biosensor communication with a measuring cell containing a protein solution. Studies were conducted for a biosensor measuring cell located along the axis of a spiral-moving liquid flow. The aggregation state of the protein was determined with an atomic force microscopy-based sensor (AFM sensor). It has been demonstrated that upon flowing of water through silicone biosensor communications, an increased aggregation of HRP protein was observed, but, at the same time, its enzymatic activity did not change. Our results obtained herein are useful in the development of models describing the influence of liquid flow in biosensor communications on the properties of enzymes and other proteins. This is particularly important for the development of serologic protein biosensors, which are beginning to be used for the early diagnosis of oncological diseases (such as brain cancer, prostate cancer, breast cancer etc.). The results obtained herein should also be taken into account when considering possible changes in hemodynamics due to increased protein aggregation.

Atomic force microscopy is employed to study the influence of the motion of a glycerol solution through a coiled (spiral-wound) polymeric communication pipe on the aggregation state of a protein, with the example of a horseradish peroxidase (HRP) enzyme. The measuring cell with the buffered solution of the protein was placed within the experimental setup over the pipe coil, through which glycerol was pumped. It is demonstrated that, in such a system, the flow of a non-aqueous liquid (glycerol) leads to a change in the physicochemical properties of a protein, whose solution was incubated in the measuring cell placed over the coil. Namely, changes in both the adsorbability onto mica and the aggregation state of the model HRP protein were observed. As glycerol-containing liquids are commonly used in biosensor operations, the results reported herein can be useful to the development of biosensor systems, in which polymeric communications are employed in sample delivery and thermal stabilization systems. The data obtained herein can also be of use for the development of specified hydrodynamic models.

In 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.

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.

Microwave radiation at 3.4–4.2 GHz frequency of the cytochrome P450 CYP102 A1 (BM3) solution was registered during the lauric acid hydroxylation reaction. The microwave radiation generation was shown to occur following the addition of electron donor NADPH to a system containing an enzyme and a substrate. The radiation occurs for the enzyme solutions with enzyme concentrations of 10−8 and 10−9 М. The microwave radiation effect elicited by the aqueous enzyme solution was observed for the first time. The results obtained can be used to elaborate a new approach to enzyme systems research, including studying of the mechanism of interaction of a functioning enzyme system with microenvironment.

Monitoring of microwave emission from aqueous solution of horseradish peroxidase (HRP) in the process of the enzyme functioning was carried out. For the monitoring, a system containing HRP, luminol and Н2О2 was employed. Microwave emission measurements were carried out in the 3.4-4.2 GHz frequency range using the active and passive modes (active-mode and passive-mode measurements). In the active mode, excitation of the solution in the pulsed electromagnetic field was accomplished. In the passive mode, no excitation was induced. It appears that the passive-mode measurements taken in the course of the peroxidase reaction in the enzyme system have shown a 0.5 °С increase of the microwave signal. Upon the active-mode measurements, taken in the same reaction conditions, the forced excitation of the solution has also led to the increase (by 2 °С) of the level of the microwave signal – i.e. to its 4-fold enhancement compared to the signal obtained in passive-mode measurements.

A combination of (atomic force microscopy) based fishing (AFM fishing) and mass spectrometry allows to capture protein molecules from solutions, concentrate and visualize them on an atomically flat sur face of the AFM chip and identify by subsequent mass spectrometric analysis. In order to increase the AFM fishing efficiency we have applied pulsed voltage with the rise time of the front of about 1 ns to the AFM chip. The AFM chip was made using a conductive material, highly oriented pyrolytic graphite (HOPG). The increased efficiency of AFM fishing has been demonstrated using detection of cytochrome b 5 protein. Selec tion of the stimulating pulse with a rise time of 1 ns, corresponding to the GHz frequency range, by the effect of intrinsic emission from water observed in this frequency range during water injection into the cell.