# Ru(terpy)-Based Conducting Polymer in Electrochemical Biosensing of Epinephrine
## 3 Results and Discussion
### Analytical Applications  
As a result of the calculated LoD and LoQ values, the Au/pRuTt/Tyr biosensor here reported does not seem suitable for the determination of EP in biological fluids, where EP is in the nmol dm\({}^{-3}\) concentration level [22]. On the other hand, the proposed biosensor can be used for pharmacological samples. For this purpose, recovery tests were performed on commercially available injectable solutions containing EP 1 mg/1 cm\({}^{3}\) (5.5 mmol dm\({}^{-3}\)) that were properly diluted to 25.0 and 50.0 umol dm\({}^{-3}\). Results reported in Table 1 were calculated as the percent ratio of the detected EP concentration to the real value and evidence a good accuracy in detecting the analyte. The RSD% (percent Relative Standard Deviation) values were calculated on five replicated measurements for each sample.  
The affinity of the enzyme to a substrate, i.e., the so-called cooperative ligand binding, is usually described by the Hill coefficient (\(n_{\text{H}}\)). This is a dimensionless parameter obtained from a curve log[I/(I\({}_{\text{max}}-\) I)] against log[C] (I = anodic current, I\({}_{\text{max}}\) = steady-state current, [C] = EP concentration (umol dm\({}^{-3}\)). In particular, when \(n_{\text{H}}\) = 1, the affinity of the enzyme for the substrate does not depend on other substrate molecules, whether bounded or not, and the kinetics of the process fit a Michaelis-Menten-type mechanism. On the other hand, when \(n_{\text{H}}\) > 1, the affinity for the substrate increases once a molecule of the substrate is  
\begin{table}
\begin{tabular}{c c c c} \hline
**EP in Samples (umol dm\({}^{-3}\))** & **EP Found (umol dm\({}^{-3}\))** & **RSD\%** & **Recovery \%** \\ \hline
25.0 & 26.9 & \(\pm\)2.14 & 107.6 \\
50.0 & 52.9 & \(\pm\)1.91 & 105.8 \\ \hline \end{tabular}
\end{table}
Table 1: Recovery tests results for Au/pRuTt/Tyr (\(n\) = 5).  
Figure 6: Effect of AA, UA, Trp, and L-cys in a 10 umol dm\({}^{-3}\) EP solution at EP:interferent ratios = 1:1 (a), 1:5 (b), 1:10 (c) in 0.1 mol dm\({}^{-3}\) phosphate buffer at the Au/pRuTt/Tyr biosensor.  
bound. \(n_{\rm H}\) values higher than 1.15 refer to a strong cooperative effect between the occupied active centers, and the Michaelis-Menten mechanism is no more adequate to describe the kinetics of the enzymatic process [23; 24]. Hence, in order to understand the kinetics of the mechanism of the biosensors studied, \(n_{\rm H}\) values were calculated and found to be 1.06 \(\pm\) 0.02 from DPV and 1.28 \(\pm\) 0.01 from CA tests, respectively. The significant difference between \(n_{\rm H}\) values calculated from the DPV and CA experiments can be reasonably ascribed to the different performing mode of these techniques: in DPV tests, the potential is continuously scanned, while in CA, a constant potential is applied to the biosensor during the entire test, causing a difference in the behavior of the biosensor as seen above in the case of the determination of LoD and LoQ. Therefore, the apparent Michaelis-Menten constant (K\({}_{\rm M}\)) can be calculated according to the adapted Lineweaver-Burk Equation (3) [24; 25] only from the DPV experiments.  
\[1/\rm I=1/I_{\rm max}+K_{\rm M}/(I_{\rm max}[C]) \tag{3}\]  
The K\({}_{\rm M}\) value was calculated as 46 \(\rm\upmu mol\) dm\({}^{-3}\), suggesting a strong affinity of the DPV biosensor towards the analyte. On the other hand, the relatively low value of I\({}_{\rm max}\) (8.3 \(\times\) 10\({}^{-7}\) A) confirms the relatively low sensitivity of the biosensor.