# Fluorescent Imprinted Nanoparticles for Sensing of Chlorogenic Acid in Coffee Extracts
## 3 Results and Discussion
### Synthesis and Characterization of Molecularly Imprinted Polymers  
fMIPs were synthesized under high dilution radical polymerization conditions following a standard protocol [25]. By optimization of solvent and monomers concentrations, it was possible to obtain polymer particles of nanometric dimensions. In this way, interactions between the template and functional monomers are favored thanks to high surface area of the particles, allowing the formation of more accessible binding sites in a short diffusion time [26]. The choice of a suitable solvent allows the formation of highly stable nano gels due to the high viscosity of the colloidal solutions. The solvating power of the solvent prevents macrogelation via osmotic repulsion forces and steric hindrance, without adding surfactants, and, furthermore, under diluted conditions in a suitable solvent, each polymeric particle is stabilized, avoiding intermolecular crosslinking [27]. To avoid aggregation and macrogelation of the polymer, it is important to keep the concentration of all monomers under a critical value, called critical monomer concentration (C\({}_{\rm M}\)). The C\({}_{\rm M}\) is defined as the percentage by weight of all monomers used for the poly merization, as compared to the percentage of the overall mass of monomers and solvent used for a polymer preparation [28]. For our purposes, DMSO was chosen as the solvent for polymer preparation due to its ability to dissolve all reagents involved in the synthesis. fMIP preparation requires three steps, which are represented in Figure 2: pre-polymerization (step I), radical polymerization (step II), and removal of the template (step III). In the pre-polymerization step, a mixture of the functional monomers and the template in DMSO was kept under stirring for 1 h at room temperature in order to favor the formation of a complex functional monomer-template. The ratio used between the functional monomer and the template was fixed at 1:1.2. A little excess of the template was used in order to obtain a high number of specific binding sites, assuming a 1:1 monomer:template interaction. The aim of this step is to favor the formation of monomer-template complexes through non-covalent bonds, allowing the pre-formation of the binding sites that will be retained after the polymerization phase. In the radical polymerization the co-monomer (if present), the crosslinker MBA or DVB and the radical initiator AIBN were added to the solution. The concentration of the crosslinker was fixed to 60%. The use of high percentages of crosslinker is essential to impart the necessary rigidity to preserve the shape and stability of the binding sites formed inside the polymeric matrix. However, polymers with a crosslinker content above 90% lead to too rigid and less flexible polymers [29]. The amount of radical initiator was fixed initially at 10% and was successively used at 5% based on the results obtained. The polymerization was carried out at 70 \({}^{\circ}\)C for 24 h.  
Figure 2: The three steps for the synthesis of fMIPs. In the last step, the removal of the template was accomplished by dialylzing the polymers in water, then in methanol:acetic acid (8:2), followed by water:methanol (90:10), and, finally, in water. The use of methanol increases the solubility of template molecules and leads to a polymer shrinkage that forces the dissociation of the MIP-template complex, while water allows one to remove the unreacted functional monomers or by-products and to resume the better polymer conformation [30]. At the beginning, the polymerization reaction, using 5-CQA as template, was conducted in DMSO-d\({}_{6}\) to verify, via \({}^{1}\)H-NMR, the possible incorporation of the template into the polymer due to the double bond present in the cinnamoyl moiety (Figure S8). In the meanwhile, it was also possible to determine the amount of unreacted monomer after polymerization using a known amount of syringic acid, which was added to the reaction mixture as the internal standard.