# Fluorescent Imprinted Nanoparticles for Sensing of Chlorogenic Acid in Coffee Extracts
## 3 Results and Discussion  
### \({}^{1}\)H-NMR Titrations  
To achieve polymers suitable for the selective identification of the analyte, it is necessary to preliminarily evaluate the interactions between the analyte and the monomers in order to establish which are the best monomers to be used. Interactions can be observed by \({}^{1}\)H-NMR titrations and the specific functionalities that are involved in the formation of the functional monomer-template complex before polymerization. It is known that the degree of template-monomer complexation is directly dependent upon the types of interactionsemployed and the chemical composition of the polymerization reaction mixture [20]. 4-Vinylpyridine (4 VP) has already been used as a functional monomer for interactions with 5-CQA and is one of the most common functional monomers used for the preparation of MIPs due to its commercial availability and low cost [21; 22]. 4-[(2-ethylenediamine)]-N-allyl1,8-naphthalimide **7** is a fluorescent molecule synthesized from 4-chloro-1,8-naphtalic anhydride [18], and its analogues have been widely used in the literature to obtain fluorescent polymers to be used in sensor development [16; 23]. The interactions between the template and each functional monomer were studied by \({}^{1}\)H-NMR in DMSO-d\({}_{6}\) by progressive addition of a solution of 5-CQA to a solution of the functional monomer in order to evaluate the variations of the chemical shift of the single protons of the monomer. In the Supplementary Material, the histograms of the chemical shift variations in the \({}^{1}\)H-NMR spectrum of 4 VP upon progressive addition of 10 equivalents of 5-CQA, as well as selected changes of NMR peaks upon titrations for the most relevant cases (Figures S3 and S4), are reported. In this way, depending on the downfield or upfield chemical shift, it is possible to identify protons involved in the interactions and the type of the interaction [24].  
The addition of 5-CQA to 4 VP solution causes a progressive shift of all signals. The variation of the proton chemical shift (\(\delta_{\rm final}\)-\(\delta_{\rm initial}\)) of the monomer 4 VP after addition of 10 equivalents of template is shown in Figure S3. Variations of the aromatic protons of the pyridine ring towards downfield are evident (Figure S4).  
In the addition of 5-CQA to the functional monomer **7**, protons of CH\({}_{2}\)NH\({}_{2}\) showed a greater shift towards downfield than the others, probably due to a proton exchange in the acid-base reaction between the amino group of **7** and the carboxylic group of 5-CQA (Figure S5). In Figure S6, it is possible to observe, specifically, the trend of the titration curve of the protons adjacent to the amino group of monomer **7**. The plateau is reached after the addition of approximately two equivalents of 5-CQA, which represents the saturation point. In order to evaluate the behavior of the template in the presence of both monomers, a \({}^{1}\)H-NMR titration was carried out by adding increasing amounts of the template to a mixture of monomers **3** and **7** (Figure S7). As it can be observed in Figure S7b, a first plateau is reached by the aromatic proton of 4 VP **3** after 0.5 eq of 5-CQA addition, but when increasing the template concentration, the chemical shift linearly increases to reach a second plateau after eight equivalents, in accordance with hydrogen bond formation between the nitrogen atom of the pyridine and the hydroxyl groups of the phenol ring. Alternatively, a \(\pi\)-\(\pi\) stacking interaction between aromatic rings could explain an upfield shift of the aromatic protons. Anyway, the main interaction of 5-CQA occurs with monomer **7**. The downfield shift of the protons CH\({}_{2}\)NH\({}_{2}\) indicates a deshielding effect due to the positively charged NH\({}_{2}\) due to the hydrogen transfer from the carboxylic group.  
Both functional monomers showed a significant interaction with 5-CQA and could contribute to the efficiency of the fMIP to recognize 5-CQA and were used in the synthesis of fMIPs.