¹ Institute of Biomedical Chemistry, Moscow, Russia

² Joint Institute for High Temperatures of the Russian Academy of Sciences, Moscow, Russia

³ Foundation of Perspective Technologies and Novations, Moscow, Russia

⁴ Moscow State Academy of Veterinary Medicine and Biotechnology Named after Skryabin, Moscow, Russia

⁵ Faculty of Computational Mathematics and Cybernetics, Moscow State University, Moscow, Russia

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.

horseradish peroxidase; enzyme aggregation; atomic force microscopy; triboelectric effect; enzyme-based biosensor

Studies on the electrokinetic phenomena associated with triboelectric effects are currently receiving a good deal of attention. This effect involves the generation of an electric charge during the frictional contact between a liquid [1,2,3,4] (or a gas [5,6]) and a solid surface upon their motion. Importantly, the motion of a gas can also lead to the triboelectric effect [5] where the electric charge that is generated induces electromagnetic fields. This effect is essential in highly sensitive biosensor systems, in which a charge generated in the analyzed solution can significantly influence its use as a measurement tool. Nanowire biosensors are extremely sensitive to an electric charge and are often utilized in typical biosensor systems [7]. It has been reported that various elements with a conical shape are employed in biosensor systems [8,9], and a number of separators have conical elements or injectors with a liquid outlet in which swirling eddy currents can form. Furthermore, rotating elements are often used for stirring the analyzed solution in a biosensor [8,10]. With regard to biosensors, the triboelectric effect must be taken into account, since it can have significant effects on the properties of the biological macromolecules that are studied with these devices [11].

Enzymes represent a type of biological macromolecules that are often used in biosensor experiments [12]. The metabolic processes in living cells are regulated by enzymes [13]. Of these, peroxidases are well represented in plant and animal tissues [13] and they play important functional roles. They catalyze the oxidation of a broad spectrum of organic and inorganic compounds by hydrogen peroxide [14]. In the human body, the role of myeloperoxidase, which is involved in atherogenesis, should be noted [15]. Horseradish peroxidase (HRP) is widely employed as a model to study peroxidases [16,17]. Moreover, it is often used in enzyme-based biosensors [18,19]. Furthermore, the use of HRP for the detection of inorganic (heavy metal) ions [20,21] and organic compounds (phenols) [22] has been reported. This protein has already been characterized in considerable detail [23,24,25,26,27,28,29], which simplifies the explanation of any experimental results obtained by using it as a model.

Many enzymes, including horseradish peroxidase, are known to form aggregates [23]. Changes in the enzyme aggregation state following external physical and chemical influences (e.g., electromagnetic, thermal, chemical influence, etc.) relate to changes in its spatial structure, which can lead to a pathological state. If this structural change does not affect the active site or chromophore groups of the enzyme, it is difficult to detect such a change by altering the kinetic parameters of the catalytic reaction. Accordingly, more sensitive methods are required for the detection of such changes in the enzyme structure. Atomic force microscopy (AFM) enables visualization at the level of single enzyme molecules [30,31,32], as was demonstrated in a study of the effect of knotted electromagnetic fields on enzyme properties [33]. Owing to its excellent height resolution [34], AFM reveals even the most subtle changes in the surface structure of visualized biological objects [35], including enzymes [36].

Herein, atomic force microscopy was employed to study the effect of a cone, rotating in air, on the aggregation state of an enzyme on mica using horseradish peroxidase as a model. To monitor the HRP adsorption properties and aggregation state after its exposure to the rotating cone, the height distribution of the adsorbed enzyme particles was plotted based on the AFM data. In addition, the HRP activity in a solution was estimated by the traditional spectrophotometric method. In our experiments, the aggregation state of HRP changed after the incubation of its solution near the apex of a rotating cone. Namely, the exposure to the rotating cone induced a shift in the height distribution maximum towards lower values, thus indicating the disaggregation of HRP. At longer incubation time, this disaggregation was accompanied by the formation of higher-order aggregates. In contrast, no effect from the stationary (non-rotating) cone was observed.

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Ivanov, Yuri D., 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, and Vadim S. Ziborov. 2022. "The Effect of a Rotating Cone on Horseradish Peroxidase Aggregation on Mica Revealed by Atomic Force Microscopy" Micromachines 13, no. 11: 1947. https://doi.org/10.3390/mi13111947