## 3 Results and Discussion
### Fluorescent MIPs  
This first series of results, although promising, was not completely satisfactory as the absolute rebinding activity of the materials is good, but not excellent in comparison with literature data on similar polymers [18]. We have therefore decided to keep 4 VP as the functional monomer while moving to fluorescent MIPs, but to change the cross-linker, choosing DVB for the last four imprinted polymers, in the hope to improve rebinding by the introduction of its further aromatic system. In the fluorescent materials, the fluorescent co-monomers **13** and **14** replaced NIPAM in the same % composition as reported in Table 1. The resulting rebinding capacity was fully satisfactory: MIPs 13-TY and 13-HT showed a rebinding of 30-35 nmol/mg, and in MIPs 14-TY and 14-HT the value was over 50 nmol/mg in both cases, although the specificity was less favorable with IF not higher than 2.7 (Table 2). Nevertheless, this value is still a good indication of the occurrence of specific sites inside the imprinted material, and sufficient to further developing sensing polymers. To this point, the fluorescent materials were designed to be used as sensors in analytical protocols to be exploited in real samples containing HT, TY and OL together, and we have therefore evaluated their selectivity in competition experiments by testing the binding capacity in solutions containing both TY and HT either 50 or 100 \(\upmu\)M (Figure 5).  
Figure 4: (**a**) Decay of TY concentration in 100 \(\upmu\)M solutions upon incubation with the MIPs; (**b**) decay of TY concentration in 100 \(\upmu\)M solutions upon incubation with the NIPs; (**c**) decay of HT concentration in 100 \(\upmu\)M solutions upon incubation with the MIPs; (**d**) Decay of HT concentration in 100 \(\upmu\)M solutions upon incubation with the NIPs.  
Figure 5: Competition tests for MIPs 13-TY, 14-TY, 13-HT and 14-HT towards the templates TY and HT.  
MIP 13-TY shows a significant selectivity at 100 \(\upmu\)M ligands towards its template during the first 60 min of incubation, after this time, selectivity decreased, and at 24 h it is actually reverted in favor of HT. MIP 13-HT is instead selective for HT even at 24 h, and its selectivity increases with the incubation time (Table 2). Additionally, MIPs 14-TY and 14-HT are selective towards their ligands, and the selectivity is constant all along the rebinding kinetics (Table 2). The selectivity is not significantly affected by the concentrations of the two ligands. In conclusion, the addition of fluorescein as the monomer and the replacement of MBA as the crosslinker with DVB enhances the possibility to have \(\uppi\)-\(\uppi\) stacking leading to a higher rebinding towards both templates although with a lower specificity and selectivity. Since our aim was the recognition of OL, which is principally present in olive leaves extract, a lower selectivity could represent an advantage. The rebinding of OL was also evaluated on MIPs 13-TY and 13-HT, since, as it will be shown in the next paragraph, they are the best performers in fluorimetry. OL is the most abundant phenolic compound in olive leaves extracts and its concentration is often 1-2 orders of magnitude higher than TY and HT. For this reason, its rebinding was studied at higher concentration, namely 250 \(\upmu\)M. Both MIP 13-TY and 13-HT performed in an excellent way, being able to remove almost completely OL from the test solution in less than one-hour incubation. This result is very important, as it confirms our starting hypothesis on the possibility of using HT and TY as templates to develop MIPs capable to rebind OL.