Molecular imprinting is a technique employed in the fabrication of biomimetic polymeric recognition matrices, possessing high substrate site selectivity and specificity. Up to now, MIPs have been prepared as bulk polymer monoliths for the selective recognition of small biomolecules (i.e., amino acids and amino acid derivatives) mainly in organic solvents. However, there is a need for the synthesis of MIPs towards larger biomolecules (i.e., peptides and protein) with well-defined physical characteristics. In the present study, epitope approach was followed for the synthesis of molecularly imprinted microparticles. According to this approach, a short peptide that represents only part of a larger peptide or protein is used as a template, which in turn can be recognized by the synthesized polymer. Conventional and inverse suspension polymerizations were employed for the synthesis of molecularly imprinted microparticles using both hydrophobic and hydrophilic oligo-peptides (i.e., Boc-Trp-Trp-Trp and Ala-Ile-Ser-Tyr-Gly-Asn-Gly-Val-Tyr) as template molecules, aiming to the purification of poly-Trp tagged proteins and class II bacteriocins containing the conserved Tyr-Gly-Asn-Gly-Val motif in their N-terminal region.

The polymerizations were carried out in a laboratory scale reactor, equipped with an impeller, an overhead condenser and a nitrogen purge line. In case of suspension polymerization, the template molecule (i.e, Boc-Trp-Trp-Trp), the functional monomer (i.e., methacrylic acid, MAA), the crosslinker (i.e., ethyleneglycol dimethacrylate, EGDMA) and the initiator (i.e., AIBN) were dissolved into chloroform. The MAA:EGDMA molar ratio was equal to 5:1 and the total amount of the monomers was equal (v/v) to the solvent used as porogen. This organic phase was added into a PVA aqueous solution (1% w/w), under mechanical stirring (600rpm) and nitrogen atmosphere. After the increase of the temperature at 60oC the polymerization was continued for 24hrs at 600rpm. The template molecule was removed from the resulted microparticles by washing with methanol-acetic acid (9:1 v/v). In case of inverse suspension, the heavy oil phase consisting of paraffin oil containing the surfactant Span-80 (2% w/v) was placed into the reactor and the steering speed was set at 500rpm. The reaction mixture was purged with nitrogen while heating to the polymerization temperature (i.e., 30^oC or 70^oC). The aqueous phase, consisting of the template (i.e., Ala-Ile-Ser-Tyr-Gly-Asn-Gly-Val-Tyr), the functional momomers (i.e., acrylic acid, AA and acrylamide, Am), the crosslinker (i.e., ethylene bisacrylamide, EBAm) and the initiator APS dissolved in distilled water, was added dropwise to the continuous phase. Polymerization was contacted for 16 hrs. When the polymerization contacted at 30^oC, 30ë TEMED dissolved in water were added to the medium after the dispersion of the monomer aqueous phase. In all cases, the dispersed aqueous phase was 2,5% v/v and the AA:Am:EBAm molar ratio was equal to 1:3:6. The produced particles were firstly washed with acetone and then with 10% acetic acid, 1% SDS aqueous solution for the template removal. Non-imprinted polymers were also produced.

As far as concerns the synthesis of MIPs towards Boc-Trp-Trp-Trp, a series of experiments were realized using different template concentrations during the polymerization, since the presence of the template at high concentration causes fragility to the synthesized microparticles, while the use of a minimum template concentration results to non specific polymer. The mean particle size of the optimum polymeric microparticles with totally spherical shape was 100ìm. The batch-wise rebinding experiments showed a higher rebinding capacity of MIPs against NIPs. The selectivity of MIPs was also proved since the MIPs rebinding towards Boc-Trp-Trp-Trp peptide was higher than the MIPs rebinding towards other hydrophobic tripeptides (i.e., Boc-Tyr-Tyr-Tyr and Boc-Phe-Phe-Phe). Additionally, the highly crosslinked poly(acrylamide) microparticles prepared by inverse suspension polymerization had particle sizes between 2-25ìm. It was shown that the rebinding capacity of MIPs against NIPs was higher either the polymerization contacted at 70^oC or at 30^oC using TEMED as catalyst. It was also shown that the thermal-inititated polymers appeared higher rebinding capacity than the polymers prepared at low temperature. Moreover, FTIR analysis revealed very few remaining un-reacted double bonds into the polymer matrix of the microparticles prepared at high temperature and no double bonds in case of the microparticles prepared at low temperature in the presence of TEMED explaining the observed difference in net binding capacity of polymers.

Acknowledgements: EU for supporting this research under the STREP Project, Proposal No. 516981.