טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentOhad Kimhi
SubjectMolecular Imprinting of Proteins
DepartmentDepartment of Biotechnology
Supervisor Full Professor Bianco-Peled Havazelet


Abstract

In recent years there is a growing interest in new materials for bioseparation, analysis, controlled drug delivery and synthetic enzymes as selective catalyst. One of the most promising fields of research that may be utilized in these applications is molecular imprinting (MI), where artificial recognition sites for a specific target molecule are created inside a matrix in a polymerization process that utilizes the target molecule as a template. Low molecular weight materials have been widely used in recent years as templates for MI . These molecules have a minimal amount of required bonds, which make them easier to imprint than large complex molecules.

This research has focused on molecular imprinting of proteins. This work presents both much needed data as well as new approaches to molecular imprinting of proteins. It is also the first time that the interactions of molecular imprinted polymers (MIP) with the template were characterized by calorimetric method (ITC) in order to get quantitative data on the adsorption process.

Two methods were used to create the MIP's, one was termed single stage polymerization and the other was termed two stage polymerization.

MIP prepared using one stage polymerization showed better affinity toward the template (lysozyme) and exhibit higher maximal capacity of the MIP compare to the control unprinted polymer. However, considerable non specific adsorption was also observed in the MIP. ITC experiments showed major changes in the adsorption enthalpy due to binding of the template molecule but very small change in the enthalpy upon binding of the reference protein (cytochrome).

Two stages MIP's were novel method introduced in order to solve the problem of non specific binding and to enhance both affinity and mechanical properties of the polymer. One procedure termed 1SMIP did not preform well but added to the understanding of the imprinting process. The second procedure termed 2SMIP was successful and exhibited lower maximal capacity for the MIP. This is the first time that such observation is made. Although the imprinted polymer exhibiting lower capacity, it showed better affinity to the template (lysozyme) and even enhanced adsorption in the presence of the competing protein. ITC measurements showed again major changes in adsorption enthalpy for the template and only minor changes for reference indicating that imprinting process did create new high affinity sites.

To sum up, the results show that imprinting process did change the matrix and created sites that have specific binding group arrangement.