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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.

The paper demonstrates a need to study the functional reliability of identification systems and the applicability of methods for ensuring the functional reliability of information systems for biometric identification systems. Methods of ensuring functional reliability of biometric identification systems via providing indicators of reliability of identification devices and introducing redundancy are considered. The applicability of the methods for biometric identification systems is investigated, its advantages and disadvantages are evaluated. Triple modular redundancy scheme has been developed for the program redundancy using N-version programming. An appropriate reliability scheme based on the principles of dual programming has been developed to implement the identification system with one additional redundant element. Based on the NIST statistics of face recognition algorithms for the developed reliability schemes the reliability indicators were evaluated and its advantages over a system consisting of a single non-redundant element have been shown. The proposed reliability schemes are consistent with the practice of ensuring the functional reliability of information systems and can be integrated into biometric identification systems that use biometric data as the main access identifier.

The paper is devoted to developing a methodology for analysing the stability of Instrumentation and Control Systems (ICS) of Nuclear Power Plants (NPPs) to the impact of computer attacks aimed at producing an accident event (targeted computer attacks). The paper demonstrates the need to consider the impact of threats to information security when analysing the safety of the process control system of the NPP power unit. Various methods of safety analysis of ICS of NPPs are considered. Their fundamental shortcomings are highlighted: the lack of consideration of the factors of anthropogenic threats to information security, dependence of failures, the complexity of presentation of modelling results with a large number of subsystems. Based on the analysis of existing practices, the requirements for the developed methodology are formed. As a result, a methodology is developed for analysing the stability of ICS of NPPs to the impact of targeted computer attacks based on the use of risk assessment matrix models: failure impact matrices and criticality matrix hierarchies. The procedure for analysing and modelling computer attacks using influence matrices and a hierarchy of criticality matrices is described. The application of the method is demonstrated by the example of the ICS segment of NPP: a scenario of a targeted computer attack is modelled using the criticality matrix hierarchy. The expediency of using failure impact matrices and a criticality matrix hierarchy for modelling targeted computer attacks on the ICS of NPPs is substantiated. The scientific novelty of the study consists in the proposal of a methodology for analysing the stability of ICS of NPPs, which takes into account the dependence of failures and allows modelling scenarios of computer attacks. Since the risk assessment matrix models are an indistinct extension of the applied Failure Mode and Effects Critical Analysis, the developed methodology can be integrated into existing practices of safety analysis of the ICS of NPPs.

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.

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.

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.

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.

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.).

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.

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.

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.

The radiothermometry (RTM) study of a cytochrome-containing system (CYP102 A1) has been conducted in order to demonstrate the applicability of RTM for monitoring changes in the functional activity of an enzyme in case of its point mutation. The study has been performed with the example of the wild-type cytochrome (WT) and its mutant type A264K. CYP102 A1 is a nanoscale protein-enzymatic system of about 10 nm in size. RTM uses a radio detector and can record the corresponding brightness temperature (Tbr) of the nanoscale enzyme solution within the 3.4–4.2 GHz frequency range during enzyme functioning. It was found that the enzymatic reaction during the lauric acid hydroxylation at the wild-type CYP102 A1 (WT) concentration of ~10−9 M is accompanied by Tbr fluctuations of ~0.5–1 °C. At the same time, no Tbr fluctuations are observed for the mutated forms of the enzyme CYP102 A1 (A264K), where one amino acid was replaced. We know that the activity of CYP102 A1 (WT) is ~4 orders of magnitude higher than that of CYP102 A1 (A264K). We therefore concluded that the disappearance of the fluctuation of Tbr CYP102 A1 (A264K) is associated with a decrease in the activity of the enzyme. This effect can be used to develop new methods for testing the activity of the enzyme that do not require additional labels and expensive equipment, in comparison with calorimetry and spectral methods. The RTM is beginning to find application in the diagnosis of oncological diseases and for the analysis of biochemical processes.

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.

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.

This study monitored thermal denaturation of albumin using microwave radiometry. Brightness Temperature, derived from Microwave Emission (BTME) of an aqueous solution of bovine serum albumin (0.1 mM) was monitored in the microwave frequency range 3.8–4.2 GHz during denaturation of this protein at a temperature of 56°C in a conical polypropylene cuvette. This method does not require fluorescent or radioactive labels. A microwave emission change of 1.5–2°C in the BTME of aqueous albumin solution was found during its denaturation, without a corresponding change in the water temperature. Radio thermometry makes it possible to monitor protein denaturation kinetics, and the resulting rate constant for albumin denaturation was 0.2 ± 0.1 min−1, which corresponds well to rate constants obtained by other methods.

Nowadays, nanobiosensors are being intensively developed due to the potential possibilities of their use for early diagnosis of diseases. This interest is enhanced by the fact that, as is known, a pathological process at an early stage is characterized by the appearance of marker proteins at very low (10−15 M and lower) concentrations in blood. Highly-sensitive nanobiosensor systems (including those based on an atomic force microscope, AFM) allows one to detect proteins at such low concentrations. The influence of the charge generated in the analyte solution flowing through the biosensor injector into the measuring cell during measurements is considered to be an important factor conditioning such a high detection sensitivity. In the present study, it was demonstrated that the presence of an AFM chip (made of mica and graphite) near the nozzle of the injector supplying an analyte solution into the measuring cell of the AFM-based fishing system (AFM-based nanobiosensors) causes an increase in charge generation upon the injection of the solution. Moreover, the influence of polymer materials (which are widely used in nanobiosensors) and communications on charge generation in the flow-based section of AFM-based nanobiosensors was studied. A stimulating influence of a low (femtomolar) concentration of proteins on the charge generation in polymeric injectors of flow-based nanobiosensors was demonstrated. Besides, a stimulating influence of an external low-frequency AC electric field on the charge generation in the nanobiosensor injector was found. Measurements were carried out in the temperature range corresponding to the physiological temperature (35 °C).

Application of micro-Raman spectroscopy for the monitoring of quality of high-k (h-k) dielectric protective layer deposition onto the surface of a nanowire (NW) chip has been demonstrated. A NW chip based on silicon-on-insulator (SOI) structures, protected with a layer of high-k dielectric ((h-k)-SOI-NW chip), has been employed for highly sensitive detection of microRNA (miRNA) associated with oncological diseases. The protective dielectric included a 2-nm-thick Al2O3 surface layer and a 8-nm-thick HfO2 layer, deposited onto a silicon SOI-NW chip. Such a chip had increased time stability upon operation in solution, as compared with an unprotected SOI-NW chip with native oxide. The (h-k)-SOI-NW biosensor has been employed for the detection of DNA oligonucleotide (oDNA), which is a synthetic analogue of miRNA-21 associated with oncological diseases. To provide biospecificity of the detection, the surface of (h-k)-SOI-NW chip was modified with oligonucleotide probe molecules (oDVA probes) complementary to the sequence of the target biomolecule. Concentration sensitivity of the (h-k)-SOI-NW biosensor at the level of DL~10−16 M has been demonstrated.