## 3 Results  
### Analysis of AFM Data  
Figure 3 displays typical AFM images (left) and corresponding cross-section profiles (right) of HRP adsorbed on mica from its solutions incubated either 1 cm below the ISP's apex (Figure 3a), 1 cm above the ISP's base (Figure 3b) or three meters away from the ISP (Figure 3c; control sample). In all the AFM images shown in Figure 3, compact objects with heights >0.5 nm are visualized on the substrate surface. These objects can be attributed to adsorbed HRP. At the same time, no such objects were visualized on the mica surface in blank experiments with protein-free buffer. Figure 4 displays \(\rho(h)\) curves (Figure 4a) and \(N_{norm}(h)\) histograms (Figure 4b) obtained after analysis of AFM data obtained in our experiments for HRP adsorbed on mica from the solutions incubated 1 cm below the ISP's apex (red), 1 cm above the ISP's base (green) or three meters away from the ISP (black; control sample). Figure 3: Typical AFM 2D (left) and 3D (right) images of HRP adsorbed on mica from its solutions incubated either 1 cm below the ISP’s apex (**a**), 1 cm above the ISP’s base (**b**) or three meters away from the ISP (**c**) (control sample). The scan size of all the images is 2 μm\(\times\) 2 μm, \(Z\) scale is from 0 to 2 nm. The \(\rho(h)\) curve obtained in the control experiments (Figure 4a, black curve) has two characteristic maxima at 1.0 \(\pm\) 0.2 nm (\(h_{max1}\)) and 1.8 \(\pm\) 0.2 nm (\(h_{max2}\)). In our previous papers, we justified that the objects of \(\sim\)1.0 nm height correspond to a mica-adsorbed monomeric form of HRP, while the objects of 1.8 nm and greater heights correspond to its aggregated form [25; 26]. In the control experiments, the \(\alpha\) ratio amounted to \(\rho_{2}/\rho_{1}\) = 0.37. In contrast to the control HRP solutions, a significant increase in the \(\alpha\) ratio was observed for the solutions incubated 1 cm below the ISP's apex (Figure 4a, red curve): \(\rho_{2}/\rho_{1}\) = 0.6. This clearly indicates an increased aggregation of the enzyme adsorbed on mica from these solutions. For the HRP solution incubated 1 cm above the ISP's base, several maxima are observed (Figure 4a, green curve). That is, while \(h_{max1}\) is still observed at 1.0 \(\pm\) 0.2 nm, \(h_{max2}\) shifts to lower heights (1.4 \(\pm\) 0.2 nm). Apart from these two peaks, a substantially broadened (1.8-3 nm) peak with the maximum shifted to 2.4 \(\pm\) 0.4 nm (\(h_{max3}\)) is observed (Figure 4a, green curve). The \(\rho(h)\) curve obtained for this solution indicates a decrease in the content of 1.0 nm-high monomeric HRP down to \(\rho(h_{max1})\) = 20%. This is in contrast to the case with the control solution, for which \(\rho(h_{max1})\) = 37% was obtained. The approximation of the experimental \(\rho(h)\) curves allowed us to determine the integral relative content of the HRP aggregates after adsorption on mica substrates in our experiments. Table 1 summarizes the results of this approximation. It should be noted that the number of mica-adsorbed objects \(N_{norm}\) normalized per 400 \(\upmu\)m\({}^{2}\) is approximately similar in all (working and control) experiments with HRP solutions (Figure 4b). \begin{table}
\begin{tabular}{c c c c} \hline \hline
**HRP Samples** & \(h_{max1}\)**, **nm** & \(h_{max2}\)**, **nm** & **\% of Oligomers** \\ \hline Below the ISP’s apex & 1.2 \(\pm\) 0.2 & 1.8 \(\pm\) 0.2 & 38 \(\pm\) 4 \\ \hline Above the ISP’s base & 1.0 \(\pm\) 0.2 & 1.4 \(\pm\) 0.2; 2.4 \(\pm\) 0.4 & 45 \(\pm\) 10 \\ \hline Control sample & 1.0 \(\pm\) 0.2 & 1.8 \(\pm\) 0.2 & 27 \(\pm\) 4 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Maximum heights of the \(\rho(h)\) curves and relative content of HRP aggregates after adsorption on mica. Figure 4: Results of processing the AFM data.