(326b) Molecularly Imprinted Polymeric Microparticles for Bio-Analytical Applications

Molecular imprinting of synthetic polymers is a process where a functional monomer and a crosslinker are co-polymerized in the presence of the target analyte (i.e., the imprint molecule) that acts as a molecular template. The functional monomers initially form a complex with the imprint molecule, and following polymerization, their functional groups are held in position by the highly crosslinked polymeric structure. Subsequent removal of the imprint molecule reveals binding sites that are complementary in size and shape to the analyte. Chemically and mechanically stable molecularly imprinted polymers (MIPs) are capable of recognizing specific substances are expected to have numerous bio-analytical applications (e.g., successfully serve as substrates for the separation of biological structures such as peptides and proteins). In the present study, suspension polymerization was employed for the synthesis of porous MIP microparticles to be used as synthetic receptors selective for the amino acid derivative boc-L-tryptophan (Boc-L-Trp). For the imprinting of Boc-L-Trp, methacrylic acid (MAA) and methacrylamide (MAm) were used as functional monomers, ethylene glycol dimethacrylate (EGDMA) and trimethylopropane trimethacrylate (TRIM) as crosslinkers, poly(vinyl alcohol) (PVA) as surfactant, azobisisobutyronitrile (AIBN) as initiator and chloroform (CHCl3) as porogen. The polymerization experiments were carried out in a laboratory scale, water-jacketed, glass reactor of 100 ml, equipped with a six-blade impeller, an overhead condenser and a nitrogen purge line. The reaction mixture was thermostated to within ?b0.05 oC with the aid of a constant temperature bath. The template molecule was removed by means of successive washing cycles with methanol-acetic acid and UV spectroscopy was employed to verify its removal. Non-imprinted polymeric (NIP) microparticles were also prepared under the same polymerization conditions. The effects of the reaction temperature, porogen concentration and type of functional monomer and crosslinker, on the morphology and porosity of the MIP/NIP microparticles were assessed by scanning electron microscopy and nitrogen adsorption measurements. It was shown that, the surface morphology and porosity of the produced particles depend strongly on the above parameters. More specifically, an increase in the reaction temperature resulted in the formation of particles with a lower specific surface area (e.g., from 360 m2/g at 60oC to 296 m2/g at 80oC). On the other hand, an increase in the porogen concentration led to an increase of the specific surface area of the produced particles. Regarding the crosslinker type, it was observed that the use of a trifunctional crosslinker (i.e., TRIM) led to the synthesis of particles with extremely low specific surface area (i.e., 45 m2/g) as compared to those prepared using the bifunctional crosslinking agent EGDMA. Finally, the type of the functional monomer and the polymer solubility in the porogen medium were found to have a direct impact on the polymer morphology. Thus, when MAm was used as functional monomer, highly porous polymeric particles with increased specific surface areas were produced (e.g., 460 m2/g). The rebinding capacity of the molecularly imprinted polymeric particles was determined by UV spectroscopy. Batchwise guest-binding experiments were performed using a small amount of microparticles incubated overnight at room temperature in CHCl3 or acetonitrile (ACN) solutions of different Boc-L-Trp concentrations. The binding capacity of the MIP microparticles was compared to that of the NIPs. Competitive analysis was also performed employing boc-D-tryptophane (Boc-D-Trp) (i.e., enantiomer of Boc-L- Trp) in order to examine the selectivity of the Boc-L-Trp imprinted polymeric receptors. It was shown, that the MIP particles adsorbed an increased quantity of the template as compared to the NIP particles and exhibited a higher affinity for Boc-L-Trp in comparison to its enantiomer Boc-D-Trp, thus proving their selectivity and specificity. Concerning the effect of the functional monomer on the rebinding of the template, the PMAm particles showed an increased binding capacity as compared to the PMAA ones. This behavior can be explained by the fact that MAm results in stronger non-covalent interactions with the functional groups of the template, which has a direct impact on the rebinding capacity of the MIP particles. The effect of the crosslinker type was also considerable. More specifically, the polymeric particles produced in the presence of TRIM had a higher backbone rigidity and consequently higher binding specificity. Regarding the porogen concentration, it was shown that an increased amount of chloroform improved the selectivity and specificity of the MIPs microparticles. Finally, when the rebinding experiments were performed in a more polar solvent (e.g., ACN), both MIP and NIP particles showed lower rebinding capacity. This means that the optimum solvent for the rebinding experiments is the porogen used in the MIPs synthesis. The present study reveals that molecularly imprinted polymeric microparticles (MIPs) can be successfully prepared by suspension polymerization using the amino acid derivative boc-L-tryptophan (Boc-L-Trp) as template. The MIP particles were found to exhibit higher affinity for the imprint molecule as compared to the NIPs and to molecules chemically related to the template, thus proving their binding specificity and selectivity and, consequently, their capability as artificial receptors for the separation of biological molecules.