## 6 Molecular Imprinting by Phase-Inversion
The composition of the p(AN-co-AA) was varied this time using the following ratios AN:AA= 80:20 and 85:15. Another method for TNT and dinitrotoluene (DNT) retention is described by [PERSON] et al. [174]. Using a phase inversion method with the introduction of the template after the polymer synthesis, a range of molecularly imprinted polymer films were prepared consisting of AN as a matrix monomer and methacrylamide (MAAm) as a functional monomer (96:4). After template removal, the films exhibited no rebinding activity, probably due to a collapse of the films' porous structure. The addition of a crosslinker (EGDMA) stabilized the macroscopic structure of the films, which provided, though limited, adsorption properties. Besides PAN copolymers, many other synthetic polymers such as polyamide [175], polystyrene and polysulfonemay be used for preparing MIMs by the phase inversion. For example, an amperometric immunosensor for the determination of antirabbit IgGwas developedusing phase inversion to prepare a porous conductor polymer graphite-polysulfone electrode [176]. Poly(ethylene-co-vinyl alcohol) copolymers (EVAL), with an ethylene molar content of 32% and 44% were used to produce MIMs, by phase inversion for the selective recognition of saccharides of biomedical interest [177]. Another polymer with great film-forming properties used for MIMs preparation is polyvinyl alcohol (PVA). However, when using PVA, chemical crosslinking is needed. In the two reports from [PERSON] and colleagues [178; 179], polyvinyl alcohol with a hydrolysis degree of over 99% and molecular weight of 1500 g/mol was used for MIMs forming and the wet membranes were crosslinked by acetylation with glutaraldehyde (GA). The templates in these two studies were the pesticides triclofon (\(O\),\(O\)-dimethyl-1-hydroxy-2,2,trichlorethyl phosphanate) [178] and atrazine (1-Chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine) [179], respectively. The procedure of crosslinking was optimized using various concentrations of GA in sulfuric acid/sodium sulfate media. The crosslinked MIMs were insoluble in water or any other solvents, thus proving the success of the crosslinking reaction. Further in the study, the authors used the remaining free aldehyde groups on the membrane surface to immobilize enzymes covalently (i.e., Tyrosinase). Thereby, these interesting studies may be useful for preparing future immuno-enzymatic electrochemical sensors. Another important advantage of the phase inversion technology is that natural (bio)polymers are not excluded, since the whole methodology is based on using previously prepared polymers [180]. An example of a biopolymer MIM prepared by DPI is provided by [PERSON] and co-workers [181], in which case a MIM was prepared in aqueous media using chitosan (CS) as the functional polymer, naringin (NG) as the template and polyethylene glycol (PEG) as the porogen. The best performance of imprinted membranes was recorded for a weight ratio of CS: NG = 15:1, when sulfuric acid was used as crosslinker. The FT-IR spectra proved that non-covalent interactionsappear between the functional polymer and the template molecules. The prepared membrane was used to separate NG from neohesperidin/NG mixtures in aqueous media. For the preparation of a hybrid natural-synthetic MIM for \(\alpha\)-amylase retention, [PERSON] et al. [182] used a mixture of dextran (Mw 76,900 Da) and poly (ethylene-co-vinyl alcohol) EVAL (intrinsic viscosity 8.44 d1/g) having an ethylene molar content of 40%. Solutions in DMSO with 15 wt% polymers were prepared with two rations of dextran: EVAL = 30:70 and 40:60, after which \(\alpha\) -amylase was added.In this study, the WPI took place in two steps, as follows: first, the casted solutions were immersed in DMSO:H\({}_{2}\)O = 50:50 solution (first phase-inversion bath) and afterwards in water (second phase-inversion bath). Interestingly, the selectivity results showed that \(\alpha\)-amylase was selectively retained by 1.96-fold compared to albumin.