## 3 Results
### Investigation of the Binding of 2-FAL to MIP by the Electrochemical SPC-MIP Sensor
The corresponding Scatchard plots (Equation (7)) for SPC-MIP6 and SPC-MIP5 are reported in Figure 5. It is seen that in the case of SPC-MIP6 only the points at high concentration, i.e., higher than 4 \(\times\) 10\({}^{-4}\) M, giving signals higher than about 30 \(\upmu\)A, are fit by the Scatchard model. At the higher concentrations K\({}_{\rm A}\) was evaluated as 4.1(3) \(\times\) 10\({}^{2}\) M\({}^{-1}\), which is similar to that found by the batch equilibration procedure in the same concentration range. From the Scatchard model the maximum signal obtainable at saturation (k\({}_{\rm CR}\), see Equation (7)) is evaluated to be 213(22) \(\upmu\)A. From Figure 4 it is seen that the corresponding NIP (SPC-NIP6) too is able to adsorb 2-FAL, but at a lower extent. Here too the concentrations which can be fit by the Scatchard model are those higher than about 2 \(\times\) 10\({}^{-4}\) M, for which the association constant is 5.4(5) \(\times\) 10\({}^{2}\) M\({}^{-1}\) not significantly different from that obtained by the sensor SPC-MIP6, indicating that the adsorption mechanism predominating at higher concentration does not involve the imprinted sites. For NIP, the maximum signal at saturation evaluated by the Scatchard plot was 75(36) \(\upmu\)A, about three times lower than that obtained by the MIP based sensor. By batch equilibration the binding of 2-FAL was the same for MIP and NIP at least up to 9 \(\times\) 10\({}^{-4}\) M. This can be ascribed to the much higher contact surface of solution and MIP in the batch equilibration experiments. In the case of the electrochemical sensor, some differences in the porosity could produce the saturation of NIP at lower adsorbed amounts of 2-FAL than MIP. A different sensor obtained by a lower amount of prepolymeric mixture, was considered too, SPC-MIP5. It was obtained by in situ polymerization of only 3 \(\upmu\)L of prepolymeric mixture. For comparison, the binding isotherm of SPC-MIP5 is reported in Figure 4 and the Scatchard plot in Figure 5.  
In Figure 5 it is seen that the SPC-MIP5 sensor, with a thinner polymeric layer, shows a typical bi-Langmuir behavior, with two kinds of sites involved in the binding of 2-FAL. One of them predominates at concentration higher than about 4 \(\times\) 10\({}^{-4}\) M (sites 1), and the other at concentrations lower than 2 \(\times\) 10\({}^{-5}\) M (sites 2). The parameters characterizing the binding of 2-FAL to the two kinds of receptors detected, obtained by the bi-Langmuir model considering the limiting slopes, are reported in Table 1:  
Figure 5: Scatchard plots for binding of 2-FAL by SPC-MIP6 and SPC-MIP5 sensors based on SWV at the following conditions: E step = 0.03 V; E pulse = 0.065 V; f = 28 pulse/s. NaCl 0.1 M water solution. The sites with lower affinity (\(K_{\rm A1}\) = 3.1 \(\times\) 10\({}^{2}\) M\({}^{-1}\)) are similar to those evaluated with the SPC-MIP6 sensor as far as the association constant is concerned, but the signal corresponding to saturation (51 \(\upmu\)A) is about 4.5 times lower than that obtained with the SPC-MIP6 sensor examined above (244 \(\upmu\)A), which was built up with an higher amount of MIP, about three times. This is roughly proportional to the amount of MIP used for the formation of the receptor layer. The association constant is similar to that obtained by the batch equilibration procedure at the same concentration level, i.e., higher than 10\({}^{-4}\) M. Actually the concentration range for which a useful signal is obtained is the same. Sensor SPC-MIP5 makes it possible to detect sites at higher affinity (3.6 \(\times\) 10\({}^{5}\) M\({}^{-1}\)) too, which are involved in 2-FAL binding at concentration lower than about 2 \(\times\) 10\({}^{-5}\) M. The higher association constant is two magnitude orders higher than that evaluated at lower concentration.