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
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.
Keywords: atomic force microscopy; peroxidase; protein aggregation; electromagnetic field; inverted pyramid; enzyme-based biosensor; molecular absorption spectroscopy
Single-molecule enzymology, which focuses on studying single enzyme molecules instead of their large ensembles, is increasing in popularity [1,2]. This approach utilizes so-called molecular detectors, which allow researchers to investigate enzymes with ultra-high sensitivity : atomic force microscopes [4,5,6,7], nanoelectronic detectors [8,9,10,11], nanopore-based sensors [12,13], total internal reflection microscopes  etc.
It should be emphasized that enzymes are now widely employed in analytical chemistry [14,15,16]. For example, horseradish peroxidase (HRP) has been employed for the detection of various heavy metal ions using microplate-based colorimetric analysis  and conventional spectrophotometry . This enzyme has also been employed for the amperometric detection of phenolic compounds . The importance of the HRP enzyme in analytical chemistry applications determined its use in our present research as a model object.
Pyramidal structures are employed in various biosensor devices [17,18,19,20]. Balezin et al.  performed a theoretical study demonstrating a change in the spatial distribution of an electromagnetic field near pyramidal structures. Moreover, these authors demonstrated that a change in the mutual orientation of a pyramid and in the direction of an incident electromagnetic field lead to a change in the topography of the spatial distribution of electromagnetic fields near the pyramid . In our recent experimental study, a change in the aggregation state of an HRP enzyme upon its adsorption on mica after the incubation of its solution near such a structure was demonstrated using atomic force microscopy (AFM) . One of the advantages of AFM consists in its visualization of the objects under study at the level of individual macromolecules . Furthermore, this is the feature that makes AFM a very useful tool in single-molecule enzymology [3,4]. Due to its extremely high, single-molecule sensitivity, AFM is capable of revealing even subtle effects of external factors on the structure of biological objects , including enzyme macromolecules . As such, by using AFM, it was demonstrated that electromagnetic fields of even a very low power density (of the order of 10−12 W/cm2) can affect physicochemical properties of enzymes .
Regarding the interaction of electromagnetic fields with pyramidal structures, on the one hand, the incubation of HRP solution near a pyramidal structure has been shown to lead to the disaggregation of the enzyme . Such an effect was ascribed to the concentration of an external electromagnetic field in certain points of space at the expense of a resonant interaction of the pyramidal structure with the field . On the other hand, not only normal but also inverted pyramidal structures have been reported to be employed in biosensor devices [17,20]. Moreover, it should be noted that the effective scattering surface of a pyramidal structure can depend on the angle of incidence of the electromagnetic waves, and, accordingly, the spatial distribution of the electromagnetic fields near this structure can depend on its spatial orientation. In this regard, it is of interest to determine whether the incubation of an enzyme solution in the vicinity of an inverted pyramidal structure has an influence on its properties.
Herein, the influence of the incubation of an HRP enzyme solution near an inverted pyramidal structure (ISP) on the enzyme’s adsorption properties was studied using AFM, while spectrophotometry was employed as a reference method. We performed these experiments in order to study whether the incubation of the enzyme solution near a pyramidal structure with different spatial orientation influences the enzyme’s properties. The incubation of the HRP solution near the ISP’s apex and above its base was found to induce an increased aggregation of the enzyme upon its adsorption on mica. The increased aggregation of HRP manifested itself in the form of an increase in both the height and lateral sizes of the AFM-visualized objects adsorbed on mica in comparison with those observed in control experiments. Here, the enzymatic activity of HRP remained unchanged in all experiments.
Since pyramidal structures of both normal and inverted orientation find their application in the construction of various biosensors, the results reported herein should be taken into account in the development of novel biosensor devices, including AFM-based systems employing pyramid-shaped tips. Our results can also be of use in the development of noise-protection systems for biosensors, including anechoic chambers with pyramidal elements.
Appl. Sci. 2022, 12(8), 4042
Ivanov, Y.D.; Tatur, V.Y.; Pleshakova, T.O.; 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. The Effect of Incubation near an Inversely Oriented Square Pyramidal Structure on Adsorption Properties of Horseradish Peroxidase. Appl. Sci. 2022, 12, 4042.