## 3 Results and Discussion
### Electrochemical Characterization of TYR-Biosen for CC Sensing  
The last approach presented here is the fabrication of a biosensor for the detection of CC, using the [Fe(CN)\({}_{6}\)]\({}^{3-/4-}\) redox probe as a mediator to infer the catalytic effect of catechol functionalities towards the electrochemistry of the previously observed [Fe(CN)\({}_{6}\)]\({}^{3-/4-}\) system (described in Section 3.5 and shown in Figure 6). To achieve this aim, SPCE modified with aminophenyl groups was chemically treated to generate aryl diazonium ions on the surface that can react with the fenic groups of the TYR enzyme by a coupling reaction, as indicated in Section 2.4.3 and depicted in Figure 1b. The characterization of the obtained surfaces in each step was carried out with the [Fe(CN)\({}_{6}\)]\({}^{3-/4-}\) system as well (as illustrated in Figure 8a). The CV of 1 mM Fe(CN)\({}_{6}\)\({}^{3-}\) in 50 mM PBS at pH 7.4 on an activated SPCE BARE (black line) shows an anodic peak of 15.26 uA at 0.193 V and a cathodic peak of \(-\)15.94 uA at 0.055 V, with a \(\Delta\)E of 138 mV (E\({}^{0/}\) = 124 mV) and i\({}_{\text{p}a}\)/i\({}_{\text{pc}}\) = 0.96. As expected, no signals of redox probe were observed on SPCE-GRAFT (red line), but they were recovered on SPCE-GRAFT-Diazo-TYR (blue line). Nevertheless, the morphology and the \"twisted\" shape of [Fe(CN)\({}_{6}\)]\({}^{3-/4-}\) voltammogram is typically obtained on enzyme electrodes, where the peak-to-peak separation is higher by 223 mV and the peak currents are 50% lower than on activated SPCE BARE with a ratio i\({}_{\text{p}a}\)/i\({}_{\text{pc}}\) = 1.04. We note that the voltammogram of the electrolyte alone on SPCE-GRAFT-Diazo-TYR (gray dashed line) does not show any peak. Figure 7: (**a**) Linear relationship between the anodic and cathodic peak currents with CC concentration in 50 mM PBS (pH 7.4) on SPCE-GRAFT-GLU-DA. (**b**) Amplification ip vs. [CC] for concentration from 0.05 mM to 1 mM. As stated above for 3 mM CC in 50 mM PBS (pH 7.4) on activated SPE BARE (red line in Figure 5a), the electrochemical behavior for 200 uM CC in the same conditions was quasi-reversible as well (red line in Figure 8b), giving rise to two observable peaks that appeared at 0.169 V with a peak current of 9.77 uA and at \(-\)0.075 V with a peak current of \(-\)10.81 uA on the forward and on the reverse scans, respectively, with a \(\Delta\)E of 244 mV (E\({}^{0\prime}\) = 47 mV) and \(\mathrm{i_{pa}}/\mathrm{i_{pc}}\) = 0.90. In contrast, an impressive catalytic current was observed for 1 mM Fe(CN)6\({}^{3-}\) on SPCE-GRAFT-Diazo-TYR in presence of 200 uM CC (black line in Figure 8b) in comparison to that obtained in the absence of CC (blue line in both Figure 8a,b), achieving an increase in the signal of about 10 times that for the CC concentration. To obtain information about the response of the TYR-biosensor towards CC in presence of the mediator (Figure 9a), the concentration dependence of CC over the range from 50 uM to 400 uM was investigated by plotting the peak current of the mediator obtained from each CC concentration (illustrated as red dots in Figure 9b). This exhibited an excellent linear correlation for the anodic peak of the [Fe(CN)6]\({}^{3-/4-}\) system that follows the linear regression equation depicted in Equation (7):  
\[\mathrm{i_{pa}}/\mathrm{\mu A}=(317\pm 7)\mathrm{[CC]}/\mathrm{mM}+(0.1\pm 0.5) ;\mathrm{R^{2}}=0.9992. \tag{7}\]  
It is worth noting that the impressive sensitivity of the TYR-biosensor was tenfold higher than the DA-sensor, as demonstrated by the values of the slopes from linear regressions, Equations (3) and (7), and as shown in Figure 9b. The developed TYR-biosensor presented a LOD and LOQ of 4.5 uM and 14.9 uM, respectively, with a RSD of 1.4% for a concentration level of 200 uM CC.