## 3 Results
### Investigation of the Binding of 2-FAL to MIP by the Electrochemical SPC-MIP Sensor  
In order to characterize other binding sites possibly present in the considered MIP, an approach based on electrochemical sensing has been considered. The device used for this investigation is schematically reported in Figure 2. The MIP layer is obtained in a very simple way by dropping a small volume of prepolymeric mixture over the whole cell and in situ thermal polymerization. The signal obtained by this technique, i.e., the peak current by SWV, depends on the concentration of the template in the polymeric receptor layer, which in turn depends on the activity in the solution phase. The binding isotherms were treated according to the Scatchard equation (Equation (7)), in order to evaluate the association constant, \(K_{\mathrm{A}}\), and the maximum signal, \(\mathrm{i_{pmax}}=\mathrm{k_{CR}}\). Sensing is based on the electrochemical activity of 2-FAL, which is reduced to furfuryl alcohol at potentials around 1-1.5 V. While this reaction is well known at the mercury electrode, it has been claimed that it does not takes place at other electrodes such as the glassy carbon (GC) and bare carbon paste electrode (CPE) [39]. In the present work, screen printed cells (SPCs) with graphite ink working electrode have been considered as the electrochemical platform for their reproducibility and low cost. It is relevant that the sensor preparation is fast and easy, since SPCs have a flat surface, in which a reproducible MIP layer can be easily deposited by drop coating, and subsequently polymerized by thermal initiation in an oven, as previously described for other analytes [36; 37; 38]. The deposition is particularly easy since it has been shown that the whole cell can be covered with MIP, with good results in terms of reproducibility and selectivity, by a simple drop coating procedure. The experimental determinations have been performed by square wave voltammetry (SWV) evaluating the peak current in function of the template concentration in the solution phase. For comparison typical voltammograms obtained at the bare screen printed cell and at MIP modified cell at different concentrations of 2-FAL are reported in Figure 3.  
The investigation of the electrochemical reduction mechanism is outside the aim of the present investigation, but it is important that an electrochemical signal depending on the concentration of 2-FAL is obtained at the screen printed cell, as seen in Figure 3.  
Figure 2: Schematic view of screen-printed cell modified by a polymeric layer, either MIP (SPC-MIP) or NIP (SPC-NIP). WE: working electrode (graphite ink); AE: auxiliary electrode (graphite ink); RE: reference electrode (silver ink). In the case of MIP-modified sensor the reduction of 2-FAL takes place at lower potential than at the bare cell, and the reduction peaks are larger. This could indicate that 2-FAL in the polymer phase is linked to sites with somewhat different affinity. Despite of the peak broadening, the peak current (i\({}_{\text{p}}\)) is related to the concentration of 2-FAL in solution, and can be used to plot the binding isotherms. Some binding curves of MIP are reported in Figure 4, together with that obtained with the corresponding non-imprinted polymer (NIP6). The binding isotherms are not straight lines but rather they present a saturation behavior at higher concentration. This suggests that binding of 2-FAL to the corresponding MIP takes place according to  
Figure 4: Binding isotherms of 2-FAL reporting the voltammetric response by SWV at the following conditions: E step = 0.03 V; E pulse = 0.065 V; f = 28 pulse/s. Sensor: SPC-MIP6 (red line), SPC-MIP5 (green line), SPC-NIP6 (magenta line). Figure 3: Voltammograms in NaCl 0.1M water solution. (**a**) bare platform, (**b**) SPC-MIP5 SWV conditions: E\({}_{\text{s}}\) = 0.01 V, E\({}_{\text{p}}\) = 0.025, f = 25 p/s. the Langmuir or bi-Langmuir model.