## 4 Discussion  
The influence of the incubation of an enzyme solution near an inverted pyramidal (ISP) structure on the enzyme adsorption properties was studied using AFM with the example of horseradish peroxidase. First, adsorption of HRP on mica from control solutions, incubated three meters away from the ISP, was studied. The number of mica-adsorbed objects, normalized per 400 \(\upmu\)m\({}^{2}\) of the substrate surface, was calculated, and density functions \(\rho(t)\) were plotted. Based on the ratio \(\alpha\) between the relative content of monomeric HRP and that of oligomeric HRP on the mica surface, the aggregation state of the enzyme in the control solutions was estimated. Second, in the same way, these data were obtained for the HRP solutions incubated either 1 cm below the ISP's apex or 1 cm above the center of the ISP's base, and they were compared with the data for the control solutions. For the HRP enzyme solution incubated for 40 min below the ISP's apex, an increased aggregation of HRP macromolecules upon the enzyme's adsorption on mica was observed in comparison with the control solution. Indeed, the relative content of oligomeric HRP increased up to 38 \(\pm\) 4% (from 27 \(\pm\) 4% in the case of the control solution; Table 1). Furthermore, the \(\alpha\) value, which characterizes the ratio between the relative content of aggregates and that of monomers, increased from 0.37 to 0.6, clearly indicating an increased enzyme aggregation. Moreover, an increase in the contribution of objects with a height of  
Figure 5: The time dependencies of the change in absorbance of 0.3 mM ABTS solution containing 1 nM HRP and 2.5 mM hydrogen peroxide. Experimental conditions: cell pathlength 1 cm, room temperature 25 \({}^{\circ}\)C.  
1.8 nm or more to the right wing of the relative distribution of adsorbed HRP with height was observed. These high objects are attributed to aggregated forms of HRP [22; 25; 26]. For the HRP solution incubated 1 cm above the ISP's base, an increased enzyme aggregation was observed: the relative content of aggregates increased up to 45 \(\pm\) 10% (Table 1). At the same time, no change in the normalized number of the mica-adsorbed objects was observed for the HRP solutions incubated below the apex or above the base of the ISP (Figure 4b). Such changes in the adsorption properties of the HRP enzyme after the incubation near the ISP can be explained by an alteration in the spatial structure of the enzyme globule. This, accordingly, leads to a change in the interaction of HRP macromolecules both with the mica substrate surface, with each other (both in the solution and on the surface) and with the water solvent. Furthermore, alterations in the structure of the HRP globule do not affect its enzymatic activity against ABTS, which remained unchanged after the incubation of its solutions near the ISP. In this regard, it must be emphasized that spectrophotometry represents a macroscopic method, in which a signal, produced by a large number of enzyme macromolecules, is recorded. At the same time, AFM operates at the level of single macromolecules. Under the action of the AFM tip, a nanomechanically assisted excitation of an enzyme can be observed [32]. The effects on enzyme aggregation, observed in our present study, are most likely caused by a rather complex process connected with the influence of external re-distributed electromagnetic radiation on the enzyme. The latter, in turn, is surrounded by ice-like water structures in the solution. These structures can change under the action of the electromagnetic radiation of various wavelengths due to a change in the ratio between para- and ortho-isomers of water in the water/enzyme system [33]. That is, the observed effect is likely caused indirectly, being connected to a change in the ratio between para- and ortho-isomers of water in the hydration shell around the enzyme globule. Accordingly, other water-dissolved enzymes can also be affected by electromagnetic radiation upon their incubation near a pyramidal structure.