November 8–9, 2017, Brno, Czech Republic 24 years MOLECULAR IMPRINTING TECHNOLOGY FOR TARGETED ANALYSIS OF PROTEINS JITKA HUTAROVA 1 , TEREZA VANECKOVA 1 , MARKETA VACULOVICOVA 1,2 , VOJTECH ADAM 1,2 1 Department of Chemistry and Biochemistry Mendel University in Brno Zemedelska 1, 613 00 Brno 2 Central European Institute of Technology Brno University of Technology Purkynova 123, 613 00 Brno CZECH REPUBLIC jithut@seznam.cz Abstract: Molecular imprinting has appeared to be an effective technique for creating of selective recognition sites in synthetic polymers. This procedure comprises polymerization of monomer in a presence of target molecules (template). The subsequent template removal forms tailor-made cavities that are complementary in shape and size to the template molecules. For protein imprinting, the choice of the suitable polymers is limited and polymerization conditions need to be optimized. In our work, dopamine monomer was chosen for polymer formation due to its nontoxicity, ease of preparation and self-assembly. For the optimization of conditions, lysozyme with molecular weight of 14.3 kDa was used and the functionality was evaluated by fluorimetry. Different concentration of dopamine and lysozyme for polymerization were tested. Under the optimized conditions, the limit of detection for lysozyme was found to be 7.8 µg/ml. Moreover, conditions for polymer formation for a purpose to reduce the overall time of analysis were investigated. The use of dopamine as a monomer in molecular imprinting shown to be beneficial in many aspects. Key Words: polydopamine, molecularly imprinted polymer, lysozyme INTRODUCTION A molecularly imprinted polymer (MIP) is a polymer with selective recognition sites (Mosbach 1994). In the procedure, a template molecule is added into a solution of suitable functional monomers (Bergmann and Peppas 2008). The most common methods of imprinting are bulk imprinting (for small template) and surface imprinting (cells or viruses); other methods are used as an alternative imprinting strategies, e.g. substructure imprinting, substructural analogues, antibody replica, or molding (Schirhagl 2014). A substantial step is template removal, which is especially challenging when imprinting macromolecules. Template can be removed by using various solvents, such as acids or bases, detergents; the polymer can be heated, or digestive enzymes (proteases) could be used. After removing of the imprinted molecule, the cavities formed in the polymer are complementary to the template in size, shape, and orientation of functionalities are left behind, and are capable to selectively recognize the target molecule (Dechtrirat et al. 2012). The optimization of the polymer structure is extremely important. The polymer should have the following properties: stiffness of the polymer structure, high flexibility, good accessibility, mechanical stability and thermal stability (Wulff 1995). Molecular recognition is a key principle in biology and bioanalysis (Dechtrirat et al. 2012). The first report about molecular imprinting for detection of protein was published in 1985, when organic silane was used as monomer for polymerization on silica beads and enzyme was entrapped (Glad et al. 1985). The following years were addressed to molecular imprinting of proteins due to the fact that proteins could not be always compatible with organic solvents used during polymer preparation (Bossi et al. 2007). Further, proteins easily subject to external influences, e.g. temperature. In the course of molecular polymerization, it is important to think of functional groups that are able to interact with functional monomer. 873