## 3 Results
### Characterization of Biosensors by Cyclic Voltammetry  
Considering the characteristics of the sensors and the spectrophotometric results regarding the activity of the enzyme, it was demonstrated that the volume of 10 \(\upmu\)L is the optimal volume of horseradish extract that can be deposited on the two screen-printed electrodes in order to obtain biosensors with improved electroanalytical characteristics, which could be used in laboratory practice. Both the SPCE/HRP biosensor and the SPCE/PB/HRP biosensor were firstly immersed in PBS pH 7 and 10\({}^{-2}\) M hydrogen peroxide solution to obtain stable responses (Figures 2 and 3) and the characteristic biosensors responses in the support electrolyte solution (Figures 2 and 3) were registered at the optimal scan rate of 0.1 V\(\cdot\)s\({}^{-1}\). Figure 1: Spectrum of the gallic acid sample with 3% hydrogen peroxide in PBS pH 7 and 5 \(\upmu\)L of horseradish extract (red line); spectrum of the 3% hydrogen peroxide in PBS pH 7 and 5 \(\upmu\)L of horseradish extract (black line); and spectrum of 5 \(\upmu\)L of horseradish extract in PBS pH 7 (green line). The cyclic voltammogram of the SPCE/HRP biosensor shows no peaks in the analyzed solution, while two pairs of redox peaks are observed for SPCE/PB/HRP. It is observed that the SPCE/PB/HRP biosensor is more sensitive than the other biosensor due to Prussian blue, which is electroactive, and the electrochemical processes taking place at the biosensor surface are shown in Equations (1) and (2) [44; 45; 46]. Figure 3: Cyclic voltammogram of SPCE/PB/HRP biosensor immersed in electrolyte support solution (\(10^{-2}\) M PBS pH 7–\(10^{-2}\) M hydrogen peroxide), (dashed line); cyclic voltammogram of SPCE/PB/HRP biosensor immersed in 30 \(\upmu\)M caffeic acid solution in electrolyte support solution (solid line). Figure 2: Cyclic voltammogram of SPCE/HRP biosensor immersed in electrolyte support solution (\(10^{-2}\) M PBS pH 7–\(10^{-2}\) M hydrogen peroxide), (dashed line); cyclic voltammogram of SPCE/HRP biosensor immersed in 30 \(\upmu\)M caffeic acid solution in electrolyte support solution (solid line). \[\mathrm{Fe}^{\mathrm{III}}[\mathrm{Fe}^{\mathrm{II}}(\mathrm{CN})_{6}]+4 \mathrm{e}^{-}+4\mathrm{K}^{+}\leftrightarrow\mathrm{K}_{2}\mathrm{Fe}^{\mathrm{II} }[\mathrm{Fe}^{\mathrm{II}}(\mathrm{CN})_{6}] \tag{1}\] \[\mathrm{Prussian\ blue} \mathrm{Prussian\ white}\] \[\mathrm{Fe}^{\mathrm{III}}[\mathrm{Fe}^{\mathrm{II}}(\mathrm{CN})_{6}]-3 \mathrm{e}^{-}+3\mathrm{Cl}^{-}\leftrightarrow\mathrm{Fe}^{\mathrm{III}}[ \mathrm{Fe}^{\mathrm{III}}(\mathrm{CN})_{6}\cdot\mathrm{Cl}]_{3}\] (2) \[\mathrm{Prussian\ blue} \mathrm{Berlin\ green}\]  
The SPCE/HRP and SPCE/PB/HRP biosensors were immersed in solutions with the same concentrations of caffeic acid, 30 uM and the cyclic voltammograms obtained are shown in Figures 2 and 3 (solid lines). As can be observed in Figures 2 and 3, in the cyclic voltammograms, anodic and cathodic peaks related to the redox processes of caffeic acid in the solution to be analyzed were observed. In the case of the SPCE/HRP biosensor, immersed in 30 uM caffeic acid solution, currents of \(-20.98\) uA at potential of \(-0.200\) V for the cathodic peak and 92.27 uA at potential of 1.100 V for the anodic peak were obtained (Figure 2). In the case of the SPCE/PB/HRP biosensor, immersed in 30 uM caffeic acid solution, current peaks of \(-41.78\) uA at the \(-0.100\) V and \(-22.4\) uA at 0.550 V for the cathodic peaks, as well as 147.5 uA at the 1.100 V and 20.2 uA at 0.190 V for the anodic peaks were observed (Figure 3). The detection mechanism of the SPCE/PB/HRP is presented in Figure 4.  
Regarding the oxidation reduction processes of caffeic acid at the sensitive element of the biosensor, it passes into its o-quinone derivative by losing two electrons and two protons and peroxidase (oxidized by hydrogen peroxide), and it reduced caffeic to its initial form by accepting two protons and two electrons [47].