# Effect of a Conical Cellulose Structure on Horseradish Peroxidase Biomacromolecules
## 3 Results  
Figure 2 shows typical AFM images of the mica substrate surface with adsorbed HRP biomacromolecules adsorbed from their solutions incubated either far away (Figure 2a; control experiment) or near the apex of the cone (Figure 2b; working experiment) for 40 min. Figure 1: Experimental setup. Numbers indicate the main elements of the setup: (**1**) is the test tube with 0.1 \(\upmu\)M HRP solution in 2 mM PBSD buffer, and (**2**) is the cellulose cone. The distance between the test tube and the cone apex is \(L=2\) cm. The AFM images in Figure 2 clearly indicate the enlargement of HRP particles on mica after the 40 min incubation of the enzyme sample near the cone apex in comparison with the control sample. In both the control and working experiments, compact objects with heights of 1 to 2 nm were observed on the surface. They can be considered HRP biomolecules, since such objects were not observed in the blank experiments. Moreover, for the samples incubated near the cone apex for 40 min, higher (up to 3 nm, Figure 2b, right panel) objects were observed. Furthermore, many of the mica-adsorbed particles were much more extended laterally in comparison with the control sample. The latter can be attributed to HRP aggregates. In contrast, at a shorter incubation time (10 min), there was no difference between the AFM images obtained for the control sample and the sample incubated near the cone. Figure 3 displays plots of the relative distribution of the AFM-imaged objects with the height _p(h)_ obtained for the samples incubated for 40 min (the distributions obtained for the shorter incubation time of 10 min did not differ from the control one and are therefore not shown). Figure 2: Typical AFM images (left) and cross-section profiles (right) of HRP biomacromolecules adsorbed onto mica. In the control experiment (**a**), the HRP sample was incubated at a \(>\)10 m distance from the cone. In the working experiment (**b**), the HRP sample was incubated at a 2 cm distance from the apex of the cone. The incubation time was 40 min. Scan size: 2 \(\upmu\)m \(\times\) 2 \(\upmu\)m. Panels on the right show cross-section profiles corresponding to the lines in the images on the left. As can be seen from Figure 3, in the working experiment, the maximum of the \(\rho\)(\(h\)) distribution corresponded to \(h_{max}\) = (1.6 \(\pm\) 0.2) nm, while in the control experiments, \(h_{max}\) = (1.0 \(\pm\) 0.2) nm was obtained. Accordingly, the 40 min incubation near the cone apex led to a considerable shift in the distribution maximum to the right in comparison with the control experiments. However, the full width at half-maximum (FWHM) in the working experiments was 0.8 nm, while in the control experiments, a 0.4 nm FWHM was obtained. The molecular weight of HRP is 40 kDa [29,30]. The AFM images of monomeric biomolecules with approximately the same molecular weight have heights of 1-1.2 nm, depending on the experimental conditions [9]. Based on this, it can be claimed that the objects of \(\sim\)1 nm height correspond to monomeric HRP, and it is this form of the enzyme that is adsorbed on the surface in both the working and the control experiments. However, in the working experiment, the distribution FWHM is much broader and, at the same time, the contribution of higher objects (which contribute to the right wing of the distribution, \(h>\) 1.4 nm) increases. The distribution width in the left wing indicates that the monomeric form of the enzyme, as well as aggregates of a higher degree of oligomerization (at which the height of objects increases), was observed. The increased contribution of objects to the right wing of the distribution in the working experiments indicates that the number of objects with a high degree of oligomerization increased in comparison with that for the control sample. The results of the AFM experiments indicate that the adsorption properties of HRP were affected after the incubation at a 2 cm distance from the cone apex for 40 min.