## 2 Molecular Imprinting Process
### Critical Aspects  
Molecular imprinting process presents various advantages, as described above, but some drawbacks must also be considered.  
Design of a new MIP system suitable for a specific template molecule often requires a lot of time and work for synthesis, washing and testing. Generally many attempts need to be made, changing various experimental parameters, before finding the optimum conditions. Combinatorial chemistry has been recently adopted in order to accelerate the optimization of MIPs to attain the desired performance [61; 62; 63]. Combinatorial approach also allows finding the best MIP composition through the simultaneous synthesis and evaluation of tens or hundreds of imprinted polymers prepared on a small scale (mini-MIPs). [PERSON] [63] introduced a high-throughput synthesis and screening (HTS) system in a 96-well plate format that allows rapid optimization and fine tuning of the molecular recognition properties of MIPs library by the use of filter plates for rapid template removal and a multifunctional plate reader for a parallel analysis of the supernatant fractions. In another example, MIP nanoparticles for peptide melittin recognition were obtained using a small combinatorial library of different functional monomers [64]. Only MIPs prepared with proper amounts of \(N\)-tert-butylacrylamide and acrylic acid showed high affinity for the peptide.  
Another important critical point is the design of water-compatible MIPs. For various applications especially in clinical and environmental fields (for instance many biomolecules are insoluble or lose their activities in organic solvents) the imprinting capability of MIPs in water solutions is required. Unfortunately water molecules will compete with the template making weaker or destroying non-covalent interactions (electrostatic, hydrogen and van der Waals bonds) between template and functional monomer. In spite of this, some progress has been made in the development of water-compatible MIPs. Hydrophobic, ionic or metal co-ordination interactions are proving to be very promising to enhance template and functional monomer association in water. Polymerizable-cyclodextrin-w were used as the hydrophobic part able to control electrostatic interactions between template and monomer from water interferences [65] or mineral oil was added after polymerization to create a hydrophobic shield tables to protect functional groups responsible for molecular recognition [66]. Moreover [PERSON] _et al_. conducted fundamental research on the physicochemical characterization of a set of imprinted and blank polymers in order to investigate why MIPs, capable of ion pairing, sometimes work in water and sometimes fail. They found that acidic monomers are more effective than basic monomers in creating ionic interactions with the template; secondly, ionic interactions character in MIP can be improved by using strong acidic monomers such as trifluoromethylacrylic acid and, finally, to enhance inclusion of ionized monomers into the polymer, the solvent should have high hydrophobicity [67]. A few studies of imprinting in a mixture of water-containing solvents (methanol/water) such as a porogen have been reported. This choice is done either for solubility reasons, when the template is a polar compound and is not very soluble in solvents, or when the use of a polar solvent favors interactions of the template with the monomers [54, 68]. Other papers reported water-compatible MIPs, prepared by introducing hydrophilic properties to the polymer in order to reduce non-specific hydrophobic interactions by using polar porogens [68], hydrophilic comonomers or cross-linkers [69] and/or monomers capable of stoichiometrically interacting with the template functionalities [70].