## 3 Results and Discussion
### Synthesis and Characterization of the Polymers  
The polymers were synthetized according to a procedure already described by us, in order to obtain micro- and nano-particles under high dilution conditions [18]. To prepare non-fluorescent polymers for the first evaluation round, monomers **5-9** were used in concentration of 1% and DMSO was chosen as the porogen solvent. NIPAM was used as the co-monomer and MBA as the cross-linker while AIBN was chosen as the radical initiator. The co-monomer was present in 15 mol % to enhance the yield of the polymerization and to enhance the stability of the polymer.  
Yields of the MIPs were in most cases satisfactory, up to 95% depending on the monomer used (Table 2). The higher yield resulted for the materials obtained from 4 VP. When 4 VP is used, together with co-monomers **13** and **14** and DVB as the cross-linker, as in the fluorescent MIPs, the yields are lower, probably due to the presence of the less reactive DVB. Among the other monomers used, 1V2P and IMID lead to the higher yields while the lower yield was for the OXO polymers. These results do not reflect the NMR observation but seem to be related to the intrinsic reactivity of the monomers. In fact, OXO is the less chemically reactive one. The corresponding non-imprinted polymers (NIPs) were also prepared in the same way, but in the absence of templates. The yields obtained for NIPs are unusually high in comparison with those of the corresponding MIPs. It is known in the literature that NIPs synthesis is very often less satisfactory as we have also verified in our experience. Nevertheless, in this case the high yield of NIPs could be explained by the absence of the template since the templates used, due to their structure, could inhibit to some extent the radical polymerization.  
TEM analysis was performed on the best performing MIPs, namely 4 VP-TT, 4 VP-HT, 13-TY and 13-HT (Figure 3). More TEM images are reported in Supplementary Materials (Figures S13-S15).  
A population of by far smaller nanoparticles can be actually observed, together with a minor fraction of aggregates. The nanoparticles observed by TEM are actually smaller than 5 nm, with very low polydispersity, with the exception of MIP 13-HT which shows an average size of 13 nm and a clearly larger polydispersity.  
\begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline \multirow{2}{*}{**MIP**} & **F.M.** & **C.M.** & **CL.** & **Yield** & **Yield** & \multirow{2}{*}{**Size b nm**} & **PDI** & \multicolumn{2}{c}{**Rebinding c** nmol/mg**} & \multirow{2}{*}{**IF c**} \\  & **15\%** & **15\%** & **70\%** & **MIP** & **NIP** & **NIP** & **TY** & **HT** & **OL** & \\  & **mol** & **mol** & **mol** & **\%** & **\%** & **\%** & **\%** & **\%** & & \\ \hline
2 VP-TY & 5 & 10 & 11 & 75 & & & & \(4.3\pm 0.2\) & & & 1.4 \\
2 VP-HT & 5 & 10 & 11 & 73 & & & (3.9 \(\pm\) 0.2) & & & (1.4) \\
4 VP-TY & 6 & 10 & 11 & 90 & & & \(4.85\pm 0.65\) & 0.135 & \(10.3\pm 0.3\) & \(2.2\pm 0.3\) & & 17.2 \\
4 VP-HT & 6 & 10 & 11 & 90 & & & \(2.40\pm 0.33\) & 0.136 & \(2.6\pm 0.1\) & \(22.0\pm 1.3\) & & 1.9 \\
1V2P-TY & 7 & 10 & 11 & 87 & & & & \(2.0\pm 0.3\) & & (13.1 \(\pm\) 1.5) & (1.1) \\
1V2P-HT & 7 & 10 & 11 & 95 & & & & \(2.0\pm 0.3\) & & 1.1 \\ IMD-TY & 8 & 10 & 11 & 96 & & & & \(4.6\pm 0.5\) & & 2.1 \\ IMD-HT & 8 & 10 & 11 & 86 & & & & \(6.6\pm 0.5\) & & 1 \\ OXO-TY & 9 & 10 & 11 & 61 & & & & \((3.0\pm 0.1)\) & (4.3) \\ OXO-HT & 9 & 10 & 11 & 50 & & & & \(0.6\pm 0.07\) & & - \\
13-TY & 6 & 13 & 12 & 74 & & & & \(0.7\pm 0.05\) & & - \\
13-HT & 6 & 13 & 12 & 80 & & \(12.97\pm 5.60\) & 0.154 & \(35\pm 2\) & & \(>\)250 & 1.5 \\
14-TY & 6 & 14 & 12 & 70 & & & \(52\pm 3\) & & \(>\)250 & 1.4 \\
14-HT & 6 & 14 & 12 & 70 & & & & \(52\pm 3\) & & 1.9 \\
14-HT & 6 & 14 & 12 & 70 & & & & \(51\pm 3\) & & 2.7 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Characterization of the imprinted materials.