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

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.).

atomic force microscopy; horseradish peroxidase; cellulose; conical structure; electromagnetic field; protein aggregation; enzyme adsorption; biosensor

The influence of electromagnetic and magnetic fields on biological systems is considered in numerous papers. This influence manifests itself not only at the macroscopic level (that is, at the level of the entire organism [1,2]), but also at the level of enzymes [2,3,4,5,6,7,8,9]. Electromagnetic fields of even relatively low power were demonstrated to affect the aggregation [9] and other properties of enzymes [5]. Data on the influence of geometric bodies of various shapes on enzyme systems in background electromagnetic fields are beginning to appear in the literature [10,11,12,13]. Namely, Balezin et al. [10] considered the calculations of changes in the spatial topography of an electromagnetic field in a pyramid, which was made of a dielectric material. These authors raised the question about the concentration of electromagnetic fields near pyramidal structures of both nanometer (and this is related to atomic force microscopy (AFM) probes) and much larger objects. Moreover, changes in the topography of background electromagnetic fields were reported to affect the physicochemical properties of enzymes incubated near pyramidal structures [9,13]. It was also demonstrated that incubation near spherical elements can also affect the properties of enzymes [12]. In this connection, the question is whether the structures of other shapes (different from pyramidal and spherical ones) influence an enzyme’s properties. It is necessary to address this problem, since it is associated with the development of enzyme-based biosensors. This issue is also important in designing specialized rooms for experiments with highly sensitive equipment. Namely, the use of conical shape elements in the construction of anechoic chambers, which are designed to suppress external electromagnetic interference when working with highly sensitive equipment [14], should be emphasized. Highly sensitive biosensors such as nanowire detectors [15,16,17] are just the case in this respect. The use of elements of conical shape in biosensors was also reported [18,19].

In our present work, the influence of a conical dielectric structure on an enzyme was studied by AFM. Owing to its ultra-high height resolution, this method allows one to reveal even subtle changes in the properties of biological objects (including enzymes) upon studying their surface [12,20] down to the level of single macromolecules [9,12,21]. For instance, AFM was successfully used to reveal the effect of weak electromagnetic fields on an enzyme [9]. A cellulose cone was used as a model of a dielectric structure. Cellulose, being the main component of wood, has unique advantages as a material for use in biosensors and energy sources, as was emphasized by Fraiwan and Choi [22]. Cellulose is an inexpensive, biodegradable and flexible material with a large surface area, thus representing an excellent solution for manufacturing biobatteries [22]. Although the use of disposable cellulose biosensors in disease detection, health monitoring and environmental pollutant detection has been reported, the ability to access an external energy source further enhances their diagnostic capabilities [23]. Moreover, cellulose is employed for electromagnetic shielding [24]. In this connection, it is important to emphasize that cellulose was reported to interact with alternating electromagnetic fields at both the colloidal [25,26] and macroscopic [25] level.

As regards food-processing applications, peroxidases are responsible for enzymatic browning, which directly affects food quality [6,7,27,28], and the effects of treatment with the use of microwave [6] and radiofrequency [7,28] radiation on these enzymes thus represent an actual phenomenon in modern food science. In our experiments, we used the horseradish peroxidase (HRP) model enzyme. This 40 to 44 kDa [29,30] enzyme glycoprotein with relatively high carbohydrate content [31,32,33] is comprehensively characterized [29,30,31,32,33,34]. This is why it represents a very convenient model in studying external impacts (such as microwave [6] and radiofrequency [7] electromagnetic radiation, or the action of gas plasma [27,35,36,37]) on peroxidase-mediated processes in food [6,7,27,35,36,37].

Herein, the effect of a conical cellulose structure on the HRP enzyme was studied at the nanoscale for the first time. In our experiments, samples of buffered HRP solution were incubated near the cellulose conical structure. After the 40 min incubation, the enzyme was adsorbed from these samples onto the surface of mica substrates, whereon they were subsequently visualized by AFM. We have demonstrated that the incubation of a solution of HRP near the apex of a cellulose conical structure for 40 min leads to a change in the enzyme aggregation state on mica. Since cellulose is known to interact with electromagnetic fields [24,25,26], the effect observed can well take place as a result of the interaction of a cellulose conical structure with the background electromagnetic field. In this respect, our study is just one step further towards the understanding of this interaction, its effects and its applications. Our findings reported herein should be considered in the development of biosensors intended for highly sensitive protein detection in order to correctly account for the changes in the properties of the studied biological macromolecules in the course of the measurements.

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Ivanov, Y.D.; Tatur, V.Y.; Shumov, I.D.; Kozlov, A.F.; Valueva, A.A.; Ivanova, I.A.; Ershova, M.O.; Ivanova, N.D.; Stepanov, I.N.; Lukyanitsa, A.A.; Ziborov, V.S. Effect of a Conical Cellulose Structure on Horseradish Peroxidase Biomacromolecules. Appl. Sci. 2022, 12, 11994. https://doi.org/10.3390/app122311994