## 3 Results
### Influence of the Caffeic Acid on the Biosensors Responses  
For the calibration of the biosensors, the signals were recorded in solution of caffeic acid with different concentrations. Figure 4: The detection principle of the Prussian blue-peroxidase based biosensor towards caffeic acid in the presence of hydrogen peroxide. R is -CH=CH-COOH. Figure 5a depicts the overlapped cyclic voltammograms of the SPCE/HRP biosensor immersed in caffeic acid solution concentrations ranging from 5 uM to 74 uM (not all are shown). Figure 5: (**a**) Overlapping cyclic voltammograms of the SPCE/HRP biosensor, immersed in different solutions with variable concentrations of caffeic acid (electrolyte support was \(10^{-2}\) M PBS pH 7-\(10^{-2}\) M hydrogen peroxide solution); (**b**) plot of linear calibration of the SPCE/HRP biosensor for caffeic acid based on anodic peak currents; and (**c**) plot of linear calibration of the SPCE/HRP biosensor for caffeic acid based on cathodic peak currents. The response of the SPCE/HRP biosensor was recorded for each concentration of caffeic acid, resulting in current values directly proportional to the substrate concentration for both the cathodic peak (at \(-\)0.100 V) and the anodic peak (at 1.100 V). After recording the response, linear regression models of the SPCE/HRP biosensor (for anodic peak, Figure 5a; and for cathodic peak, Figure 5b) were obtained for the detection of caffeic acid from the dependence between the intensity of the peak current and caffeic acid concentration. From the slope of the dependence between the current and the concentration of caffeic acid in the solution, the detection and quantification limits of the biosensor were calculated. The standard deviation for the blank sample was calculated from seven replicates of cyclic voltammograms recorded in an electrolyte solution containing no caffeic acid. The sensitivity of the SPCE/HRP biosensor based on anodic peak variation is 0.17 uA\(\cdot\)uM, indicating an adequate performance for the detection of caffeic acid. For the anodic peak, the limit of detection (LOD) is 3.86 uM and the limit of quantification (LOQ) is 12.87 uM, calculated according to Equations (3) and (4). \[\text{LOD}=3\,\times\,\sigma/\text{m} \tag{3}\]  
\[\text{LOQ}=10\,\times\,\sigma/\text{m} \tag{4}\]  
where \(\sigma\) is the standard deviation of the biosensor signal in the blank sample and m is the slope of the calibration linear equation [49]. For the cathodic peak, the limit of detection (LOD) is 5.58 uM and the limit of quantification (LOQ) is 18.60 uM  
Figure 6a shows the overlaid cyclic voltammograms of the SPCE/PB/HRP biosensor immersed in \(10^{-2}\) M PBS pH 7-\(10^{-2}\) M hydrogen peroxide solution and caffeic acid solutions in a concentration range from 5 uM to 74 uM (not all are shown). The response of the SPCE/PB/HRP biosensor in caffeic acid solutions of different concentrations was recorded, resulting in current values directly proportional to the substrate concentration for both the cathodic peak (at E of \(-\)0.100 V) and the anodic peak (at E of 1.100 V). From the cyclic voltammograms, calibration linear models of the SPCE/PB/HRP biosensor (for both peaks--Figure 6b,c) were made for the detection of caffeic acid, taking into account the current intensities as a function of the concentration of caffeic acid in the solution, and the detection and quantification limits of the biosensor were calculated. The sensitivity of the SPCE/PB/HRP biosensor taking into account the anodic peak increment when the concentration increases is 0.72 uA\(\cdot\)uM\({}^{-1}\). This value is four times higher compared with the sensitivity obtained for the SPCE/HRP biosensor. This fact demonstrates the importance of the PB from the sensitive layer as the electron mediator. For the anodic peak, the limit of detection (LOD) is 0.90 uM and the limit of quantification (LOQ) is 3.00 uM. For the cathodic peak, the limit of detection (LOD) is 1.96 uM, and the limit of quantification (LOQ) is 6.53 uM.