## 2 Molecular Imprinting Process
### Prepolymerization Studies  
Good interactions between monomer and template represent a preliminary essential condition to obtain MIP networks with potential recognition sites. Today there are two main strategies employed for MIP technology depending on the nature of prepolymerization interactions between template and monomer:  
* self-assembling approach [35], similar to the biological recognition systems, that uses non-covalent forces, such as hydrogen bonds, Van der Waals forces, ion or hydrophobic interaction and metal-coordinations;* preorganized approach [2], which uses covalent reversible bonds giving a rather homogeneous population of binding sites and reducing the non-specific sites. However, to remove the template from the polymer matrix, it is necessary to cleave the covalent bounds. Amongst these, self-assembling is still the most frequently adopted approach for the preparation of MIPs due to the simplicity of complex formation and dissociation and the flexibility in terms of available functional monomers that can interact with almost any kind of templates. On the other side, the related MIPs have lower binding affinity than those prepared using covalent methods. [PERSON] and [PERSON] [36] prepared, by using a bulk technique, cholesterol-imprinted polymers either in a covalent and a non-covalent approach and they compared MIPs performances as stationary phases for HPLC columns. They found less peaks broadening, higher adsorption capacity and about fivefold higher chromatographic efficiency for the covalently imprinted polymer in comparison with non-covalently imprinted polymers. Various works reported in the literature demonstrated that prepolymerization studies on self-assembling systems can be useful for the selection of suitable functional monomers and solvents for specific template molecules. The formation of functional monomer-template complexes has been investigated by spectroscopic and theoretical approaches, that includes UV-Vis and NMR spectroscopies and theoretical models as well [37; 38; 39; 40; 41; 42; 43]. [PERSON]'s method, titration curves and binding isotherms are currently used to determine the nature of the interactions, the coordination number of the complex monomer-template and the association constant by using spectroscopy techniques. For example, the formation of the complex between nitrofuranotin with 2,6-bis(methaacrylamide) pyridine (BMP) was studied by NMR spectroscopy revealing a hydrogen bonding interaction between the imide moiety of nitrofuranotin with the protons of the pyridine moiety of BMP and allowing also the calculation of the association constant. Similar studies were also reported between carboxyphenyl aminohydantoin (CPAH) and 2,6-bis(methacrylamide) pyridine (BMP). Titration curves were performed and the binding constant was calculated using nonlinear curve fitting for a 1:1 complexation model and the complex stoichiometry was independently confirmed by a Job plot method [44]. [PERSON] _et al._ in 2006 [45] evaluated the binding affinity and selectivity of a new phthalocyanine, as potential monomer towards nucleoside derivatives, by using UV-vis titration experiments. The experiment allowed the calculation of the association constant Ka, determined by the modified Benesi-Hildebrand equation, of a zinc phthalocyanine with tri-\(O\)-acetyladenosine (TOAA). The calculated value was 1.35 \(\times\) 10\({}^{4}\) M which confirms potential use of the zinc phthalocyanine as functional monomer in the formation of molecularly imprinted polymers for nucleoside receptors. Successively, [PERSON] _et al._[27; 34] prepared selective MIPs having the phthalocyanine-based recognition centre as receptors for tri-\(O\)-acetyladenosine (TOAA) or for RNA nucleoside (Figure 2). In 2009 [PERSON] _et al._[40] reported the studies of prepolymerization interactions between nicotinamide and methacrylic acid in chloroform and acetonitrile by using \({}^{1}\)H-NMR spectroscopy.