## 2 Molecular Imprinting Process
### Optimization of MIPs Synthesis
The cross-linker is important in controlling the morphology of the polymer matrix, serves to stabilize the imprinted binding sites and imparts mechanical stability to the polymer matrix in order to retain its molecular recognition capability [49]. Different cross-linkers have been used (Figure 5). High cross-link ratios are generally used in order to access permanently porous (macroporous) materials with adequate mechanical stability. Ethylene glycol dimethacrylate (EGDMA) and trimethylolpropane trimethacrylate (TRIM) are the most commonly employed. Some authors found that cross-linker has a major impact on the physical characteristics of the polymers and much less effect on the specific interactions between the template and functional monomers [37, 50, 51]. TRIM as cross-linker gives polymers with  
Figure 4: Structure of the most common monomers used for molecular imprinting. more rigidity, structure order and effective binding sites than EgDMA. In the case of polymerization obtained by precipitation method, it has been seen that optimizing the amount of cross-linker and reducing the concentration of the template, the polymer binding properties are improved and the level of non-specific interactions is decreased [52]. In another study it has been observed that the type of cross-linker strongly influences the final size and yield of MIP nanoparticles [53]. In fact, when divinylbenzene was used as the cross-linker, polydisperse MIP particles were obtained in low yield, whereas, trimethylolpropane trimethacrylate (TRIM) led to uniform nanoparticles in high yield (90%). Moreover, nature and volume of the solvent play also an important role in the molecular imprinting process. The most common solvents used for MIPs synthesis are toluene, chloroform, dichloromethane or acetonitrile. The solvent serves to bring all the components (monomer, template, initiator, cross-linker) into one phase in the polymerization and it is responsible for creating the pores in macroporous polymers. The solvent should produce large pores to assure good flow through properties of the resultant MIP and increasing the volume of the solvents, the pore volume of the polymer enlarges. For this reason it is quite common to refer to the solvent as the \"porogen\". The porogen in a non-covalent imprinting polymerization should also be chosen considering its role in promoting template-functional monomer complex formation: less polar solvents such as chloroform or toluene increase the complex formation facilitating polar non-covalent interactions such as hydrogen bonds; more polar solvents tend to dissociate the non-covalent interactions in the prepolymer complex, especially protic solvents that afford a high degree of disruption of the hydrogen bonds. However in some papers efficient MIPs have been prepared in rather polar solvents (e.g., acetonitrile/water or methanol/water) since strong template-monomer interactions have been observed. For instance, a water-compatible imprinted polymer with strong affinity for a polar 1-methyladenosine template has been prepared in acetonitrile/water 4/1 and successfully used as solid phase extraction (SPE) sorbent for the analyte extraction from spiked human urine samples [54]. Usually apolar, non-protic solvents, such as toluene or chloroform, are preferred in MIPs synthesis whereas, if hydrophobic forces are involved in the complexation process, water or other protic solvents  
Figure 5: Structure of the most common cross-linkers used for molecular imprinting. could be selected as well. Moreover, it was observed that, after the polymerization, the rebinding performance is optimized when carried out in the same solvent used for the imprinting, suggesting that the optimum rebinding conditions require the same or very similar solvation conditions used for the polymerization.