## 3 Results and Discussion
### Fluorimetry  
The promising properties measured in the rebinding tests were confirmed by fluorimetry. The pH of the aqueous extracts of olive leaves as obtained by our protocol is 5.6, and we have measured an average conductivity of 3.6 mS/cm, similar to that usually found in plant aqueous extracts and in physiological buffers. The pH of a colloidal solution of our fluorescent MIPs at 2.5 \(\upmu\)g/mL in water is 6.6. To take into account potential effects of pH and ionic strength, the polymers were tested under different conditions, namely in water as 2.5 \(\upmu\)g/mL colloidal solutions, in 100 mM citrate buffer at pH 5 (below that of real samples and at similar ionic strength), and in PBS buffer at pH 7.5 (a value over the pH of real samples and over the third pKa of fluorescein). TY, HT and OL were added to the MIP in a range of concentrations spanning from 10 fM to 100 \(\upmu\)M. The amount of MIP was nearly 3-orders of magnitude less than that used in the rebinding assay, as the fluorescence emission was strong enough to allow working at very low concentrations. Under these conditions, a 10 min incubation time was sufficient to obtain stable changes in emission. Fluorescence was monitored at 480 nm excitation and 515 nm emission and, as expected, it did increase in citrate buffer, and in water. MIPs 13-TY and 13-HT resulted much more sensitive, and their response curves in water are reported in Figure 6. The emission spectrum of MIP 13-TY upon titration with TY is reported in Figure 6 as an example, showing the increase of the emission peak, without changes of its shape and drifts of the emission maximum, thus supporting the evidence that the phenomenon is related to a true activation of the emission. The same was observed also in all the other titrations. The overall change is nevertheless larger in water at pH 6.6 than in citrate buffer at pH 5. Conversely, only a negligible change was observed when the titrations were carried out at pH 7.5 in PBS buffer, where the initial emission of the MIPs is much higher (Supplementary Materials, Figures S16 and S17). The whole change occurs at very low concentration of phenols, in a range between 100 fM and 100 nM. This ensures that it is not influenced by changes in pH, that are actually not detectable also in water after the addition of 100 nM targets. Before showing in the last section the potential of such system for the detection of our phenols, we discuss the possible origin of the activation of fluorescein emission. At pH 7.5, the dianion of fluorescein predominates in solution, representing, on the basis of the pK\({}_{\text{a}}\), about the 83% of the total fluorophore. The dianion is more fluorescent than the monoanion, which is the most abundant species at both pH 6.6 (84%) and 5 (77%). As no changes in emission are observed at pH 7.5, we can exclude that the activation is due to an interaction of the phenols with the dianion. The monoanion seems to be rather involved, and the interaction with the phenols inside the MIP could actually shift the lactone-quinone equilibrium of the monoanion towards the fluorescent quinone form, as envisaged in our starting hypothesis, or, more unlikely, could shift the equilibrium monoanion/dianion towards the dianion. Both the polymers are sensitive to the whole set of tested ligands. In this evaluation, MIP 13-TY is more selective towards its template TY, while HT and OL give a very similar response. Although the absolute increase of signal is higher in 13-TY, also 13-HT performs well and the sensitivity is very high. It is less selective than 13-TY, as it could be expected from the structure of its ligand, and the interactions with the three phenols leads to similar results. The titration curves of Figure 6 could be in principle exploited as calibration curves for the detection of phenols.