# Ru(terpy)-Based Conducting Polymer in Electrochemical Biosensing of Epinephrine
## 3 Results and Discussion
### Determination of EP with a Au/pRuTt/Tyr Biosensor  
An efficient immobilization of the enzyme on an electrode surface is the most critical step in biosensors development. In this context, the design of the polymer film here proposed includes the presence of \(-\)COOH groups with the aim to induce a chemical bond between the polymer-modified electrode surface and the enzyme. The use of glutaraldehyde as a coupling reagent between enzyme molecules in a classical cross-linking reaction further contributes to the immobilization of the enzyme [17]. In this perspective, Tyr was firstly deposited on the gold or glassy carbon electrode surface modified by the pRuT film and then immobilized with the help of glutaraldehyde (as described in Section 2.3). The electrochemical behavior of the Au/pRuTt/Tyr biosensor was preliminarily investigated by cyclic voltammetry. The CV response of a 550 \(\mu\)mol dm\({}^{-3}\) of EP solution in the phosphate buffer evidences a broad and ill-resolved oxidation process at about 0.2 V (Figure S2). In order to better ascertain the analytical behavior of the reported biosensor, DPV tests were performed between \(-\)0.6 and 0.6 V in an EP concentration range between 1 and 100 \(\mu\)mol dm\({}^{-3}\). The DPV responses show two peaks (Ep = \(-\)0.18 and 0.16 V, respectively), with the more anodic one being significantly more intense (Figure 3 Left). These peaks are reasonably attributable to two different oxidation processes: from EP to adrenalinequinone (an open-chain quinone structure) and from the cyclized form of this quinone to adrenochrome [18; 19; 20]. Calibration tests were performed on the more anodic (and more intense and sharper) peak, evidencing a linear increase in the current at an increasing EP concentration in the whole range considered (Figure 3 Right) with a good linear correlation (R\({}^{2}\) = 0.998). The DPV responses at Au/pRuTt electrode in the same EP  
Figure 2: Cyclic voltammetry characterization of a pRuTt film on a gold disk electrode in CH\({}_{3}\)CN/0.1 mol dm\({}^{-3}\) TEAPF\({}_{6}\) solvent system, Q\({}_{\text{dep}}\) = 32 mC cm\({}^{-2}\) (blue line). The cyclic voltammetry (CV) curve of the gold disk electrode in the same conditions is reported for comparison (red line). concentration range (Figure S3) show a decrease of an order of magnitude in the current intensity, confirming the electrocatalytic effect due to the presence of the enzyme. Limit of detection (LoD) was calculated according to Equation (1) [21]:  
\[\text{LoD}=3.29\sigma_{B}/b \tag{1}\]  
where \(\sigma_{B}\) is the standard deviation of the blank response (calculated on 11 replicates of the blank), and \(b\) is the slope of the regression line. LoD was calculated to be 0.67 umol dm\({}^{-3}\). Analogously, limit of quantification (LoQ) was calculated according to Equation (2)  
\[\text{LoQ}=5\sigma_{B}/b \tag{2}\]  
and was found to be 1.02 umol dm\({}^{-3}\). Finally, sensitivity, calculated as the ratio of the slope of the calibration curve to the area of the electrode surface, was 3.1\(\cdot\)10\({}^{-7}\) A umol\({}^{-1}\) dm\({}^{3}\) cm\({}^{-2}\). The analytical behavior of the Au/pRuTt/Tyr biosensor was also investigated by chronoamperometry (Figure 4), and the results were compared to those obtained by DPV. The working potential was chosen at 0.18 V, according to the DPV response, and a concentration range between 1 umol dm\({}^{-3}\) and 1 mmol dm\({}^{-3}\) was investigated. An increase in current at the increasing EP concentration was observed with a good linear correlation in the range 1-100 umol dm\({}^{-3}\) (R\({}^{2}\) = 0.998). Figure 4: (**a**) Current/time responses of the Au/pRuTt/Tyr biosensor at 0.18 V in 0.1 mol dm\({}^{-3}\) phosphate buffer in the EP concentration range 1 μmol dm\({}^{-3}\div\) 1 mmol dm\({}^{-3}\) (main graph) and 1 μmol dm\({}^{-3}\div\) 100 μmol dm\({}^{-3}\) (inset). (**b**) Calibration curve in the EP concentration range 1 μmol dm\({}^{-3}\div\) 100 μmol dm\({}^{-3}\).