# Fluorescent Imprinted Nanoparticles for Sensing of Chlorogenic Acid in Coffee Extracts
## 3 Results and Discussion
### Fluorescence Titrations  
The fluorescent properties of polymer fMIP01 were evaluated by studying the interactions between fMIP01 and the target molecule (5-CQA). Changes in the polymer fluorescence at low concentrations were registered by fluorescence titration of the polymer, with increasing amounts of the analyte in two different solvents: DMSO and water:DMSO (9:1). As a first choice, titrations were carried out by adding increasing amounts of 5-CQA from micromolar to millimolar concentrations to a solution of 30 \(\upmu\)g/mL of fMIP01 in DMSO. In Figure 8, we can observe a three-fold increase in fluorescent intensity at the emission maximum of 530 nm by addition of the analyte. The fluorescence enhancement is probably due to the hydrogen transfer between the carboxylic group of the analyte and the amino group of the monomer 7 present in fMIP01, making PET (photoinduced electron transfer) communication between the receptor and the fluorophore not thermodinamically favored, and it becomes cut off, since the lone pair of electrons of the receptor are no longer available after recognition of the analyte. As it has been reported in the literature, in these types of \"off-on\" systems, that protons from the analyte electrostatically attract the electrons, thus increasing the oxidation potential of the analyte-bound receptor [(33; 34)]. Figure 6: HPLC chromatograms of coffee extract (**a**) UV at 325 nm, peak 1: 5-CQA; (**b**) UV at 270 nm, peak 1: 5-CQA, peak 2: caffeine. Figure 7: (**a**) Rebinding kinetics of 5-CQA and caffeine in coffee extract; (**b**) HPLC chromatogram of coffee extract with fMIP01, UV at 270 nm, peak 1: 5-CQA, peak 2: caffeine. Changing the solvent from DMSO to a mixture of H\({}_{2}\)O/DMSO 9:1 (at a pH of 7.7), a bathochromic shift with respect to the system in DMSO was observed, with the emission maxima registered at 540 nm instead of 530 nm. This effect could be a consequence of the \(\pi\)\(\rightarrow\)\(\pi^{\circ}\) internal electron transfer transitions (ITC) that have already been observed for these types of dyes when the polarity of the solvent increases [18]. Moreover, a quenching of the fluorescence was instead observed (Figure 9), showing a decrease in fluorescence of 58% by adding a 39 \(\upmu\)M solution of the analyte, while a total quenching of 92% was obtained at a final concentration of 80 mM of the target molecule. A possible explanation for the quenching is that, in presence of water, the amino group of the fluorescent monomer can be protonated, cutting off the PET process, which means that the system has been switched \"on\" prior to the recognition event. The Stern-Volmer plot obtained with fMIP01 is reported in Figure 10, where a bimodal quenching behavior can be noticed with a first, higher slope, linear region at low concentrations of 5-CQA and a second, lower slope, linear region at higher 5-CQA concentrations. This behavior can be an indication of the presence of non-homogeneous binding sites inside the polymers with different affinity for 5-CQA. Figure 8: Fluorescent emission titration spectra of fMIP01 in DMSO after addition of 5-CQA. Figure 10: Stern-Volmer plot of fMIP01 for 5-CQA. Figure 9: Fluorescent emission titration spectra of fMIP01 in water:DMSO (9:1) after addition of 5-CQA. The efficiency of the analyte in quenching the fMIP01 emission was investigated by the Stern-Volmer Equation (4), [15] which was separately applied on the two linear regions of the Stern-Volmer plot (Figure 10). \[\frac{F_{0}}{F}=K_{app}^{SV}\cdot[5-CQA]+1 \tag{4}\]  
The bimolecular quenching constant \(K_{q}^{app}\) can be calculated by Equation (5) [35]:  
\[K_{q}^{app}=\frac{K_{SV}^{app}}{\tau_{0}} \tag{5}\]  
\(K_{q}^{app}\) was calculated using the lifetime (\(\tau_{0}\)) of an analogue 4-amino substituted napthalimide monomer used to develop fMIPs to respond to carboxylate-containing guests. The reported \(\tau_{0}\) of this molecule in DMSO corresponds to 0.11 ns [36].