# Fluorescent Imprinted Nanoparticles for Sensing of Chlorogenic Acid in Coffee Extracts
## 3 Results and Discussion
### Immobilization of fMIP01  
Since physical entrapment of MIPs into gels or membranes has been previously reported in the literature for their use as electrochemical transducer, [38] the fluorescent polymer fMIP01 has been physically entrapped into a gelatin gel covalently bonded to a functionalized support. The entrapment has been carried out using a suspension of 100 \(\mu\)g/mL of fMIP01 in water:DMSO (9:1) and a solution of 20 mg/mL of gelatin, which were mixed together in a ratio of 1:1 and immobilized on a poly-lysine glass support using glutaraldehyde (GA) as a bifunctional crosslinking agent (Figure 11). Gelatin is a bio-macromolecule obtained by the hydrolysis of collagen, mostly from skin and the connective tissue of animals. Because of the gel-forming property of gelatin at around 35 \({}^{\circ}\)C, and its versatility in the amino acid composition, it has been widely used, not only as a stabilizer, emulsifier, and clarifying agent, but also as a protective  
Figure 11: Schematic representation of fMIP immobilization. coating material [39; 40]. Moreover, due to its low cost and efficiency in the stabilization of collagenous materials, GA is the most widely used cross-linking molecule able to form a Shiff base by reaction of non-protonated amino groups (-NH\({}_{2}\)) of lysine to the carbonyl groups (C=0) of the aldehyde. The mechanism of the covalent bond formation between the gelatin and the GA is greatly dependent upon pH conditions, and it has been extensively reported in the literature [39]. The new covalent bonds can be either intramolecular (short-range) or intramolecular (long-range), which are the result of the polymerization of glutaraldehyde or alidol condensation reaction. The residual immobilized concentration of the fluorescent polymer was estimated by calculating the ratio between the initial fluorescence intensity and the fluorescence intensity after washing the glass plates with water at 540 nm of different polymers solutions immobilized on fifteen different poly-lysine plates in different times. Mean values are reported in Table 4 and, as can be noticed, about 70% of the initial polymer concentration remains on the plates. Once the polymer was immobilized, the fluorescence properties of the polymer were evaluated by carrying out titrations, in duplicate, with increasing concentrations between 78 \(\upmu\)M and 80 mM of a standard solution of 5-CQA. The fluorescence emission of the fMIP01 was measured at 540 nm when excited at 480 nm, before and after incubation, for 90 min. The fMIP01 fluorescence resulted in a correlated quenching in a range from 156 \(\upmu\)M to 40 mM of the analyte (Figure 12). From the slopes of the equation in the Stern-Volmer plot at high and low concentrations, the apparent quenching constants were calculated, as described in the previous section, and the obtained values are 3.5 and 8.6 \(\times\) 10\({}^{11}\) L-mol\({}^{-1}\)s\({}^{-1}\), respectively. Although these values indicate that a static quenching is taking place, the significantly lower values of the apparent Stern-Volmer constants (39 and 91 L-mol\({}^{-1}\)), with respect to the ones obtained by carrying out the titrations in solution (2.11 \(\times\) 10\({}^{2}\) and 1.6 \(\times\) 10\({}^{3}\) L-mol\({}^{-1}\)), indicate a \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Initial MIP** & **F intensity before** & **F Intensity after** & **Imobilized MIP** \\
**Concentration** & **Washing (STD)** & **Washing (STD)** & **Concentration** \\  & & & **(ug/mL)** \\ \hline
100 & 39,453.17 (6437.04) & 27,941 (5766) & 71 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Fluorescent intensities of immobilized MIP01. Figure 12: Preliminary calibration curve of immobilized MIP01 with 5-CQA (**left**), Stern-Volmer plots of immobilized fMIP01 for 5-CQA (**right**, green line: regression of low concentration data; magenta line: regression of high concentration data). lower affinity of the polymer for 5-CQA.