## 4 Discussion  
Adsorption isotherms in different concentration ranges have been obtained by the different characterization techniques proposed in the present investigation. For comparison, the binding isotherms of the same MIP with the different methods are reported in Figure 10 using the log-log relationship [41; 42] in order to clearly compare the whole considered concentration range from \(10^{-7}\) to \(10^{-2}\) M. The depending variables are different, according to the different method used and as specified in the legend.  
Only in the case of the batch equilibration method a straight line with slope only slightly lower than 1 is obtained, indicating that conditions far from the saturation [19; 20; 29] are considered in the reported experiments. Notice that in this case the ratio of the solution volume to the mass of adsorbing MIP is \(75\) mL\({}_{\text{ext}}\)/g\({}_{\text{MIP}}\). Moreover, the Scatchard plot (Equation (3)) shows the presence of only one kind of binding sites, whose association constant and concentration (c\({}_{\text{R}}\)) have been evaluated, as reported above. These results do not imply that other classes at higher affinity are not present in the considered MIP, but only that, if present, they are at lower amount than the non-specific sites.  
On the contrary, a saturation is clearly observed in the binding isotherm by electrochemical sensor at concentrations higher than about \(3\times 10^{-4}\) M (see Figure 4). The difference can be ascribed to the fact that in this case the ratio of the solution volume to the mass of adsorbing MIP is about \(1250\) mL\({}_{\text{sol}}\)/g\({}_{\text{MIP}}\), i.e., much higher than that in the batch experiment. At lower concentration a behavior approaching saturation is observed too, since the log-log isotherm in the lower concentration range has a slope well below 1. This indicates the existence of at least two kinds of binding sites, so that the bi-Langmuir model has been applied to evaluate the respective affinities as reported in Table 1. It is relevant that the same affinity, indicated as K\({}_{\text{A}}\) in batch equilibration and K\({}_{\text{A1}}\) in electrochemical sensing, is evaluated in the same concentration range, using completely different investigation methods and conditions.  
Figure 10: Adsorption isotherms obtained by different methods: optical SPR-MIP sensor, electrochemical SPC-MIP sensor and by batch equilibration and spectrophotometric analysis.  
Due to the better sensitivity and reproducibility of the electrochemical sensing compared to the batch equilibration, other imprinted sites with higher affinity for 2-FAL are detected too, and characterized by their association constant, K\({}_{\text{A2}}\). It is worth noticing that this part of the log-log binding isotherm has a very low slope, which indicates that the detected sites are near to the saturation (see the k\({}_{\text{CR2}}\) value in Table 1). The D-shaped POF-MIP-SPR sensor responds to even lower concentrations than the electrochemical one, partially overlapping the concentration range detected by the electrochemical sensor. As in that case, both the log-log isotherm (Figure 10) and the Scatchard plot (Figure 9) reveals the presence of two different kinds of sites for 2-FAL, one of which, that detected in the same concentration range, is very similar to that determined by the electrochemical sensor. In addition, the SPR sensor allowed to investigate a lower concentration range, corresponding to sites with even higher association constant, K\({}_{\text{A3}}\) (see Table 2).