Development of Functional Protein-based Target-specific Labeling Nanoplatforms for Biological Applications

Abstract

Qualitative and quantitative analyses of target biomolecules in the cell are essential for the early detection of diseases and the prognosis of treatment in various biomedical research and clinical applications. To selectively detect and quantify biomolecules of interest in complicated biological samples, various methods have been developed in the biotechnology fields. Target biomolecules in the cells can be specifically visualized with labeling probes by fluorescent cell imaging and quantified with immunoassay based on antigen-antibody interactions.

The aim of the thesis is to develop the functional protein-based target-specific labeling nanoplatforms for application in fluorescent cell imaging and immunoassays. Target biomolecules within the cell can be detected with target-specific fluorescent cell imaging probes. Protein cage nanoparticles as attractive polyvalent nanoplatforms have been applied to bioimaging probes and biosensor components because they have a well-defined symmetric hollow shell structure with uniform nanoscale particle sizes. Their polyvalent nature allows uniform multiple targeting ligands or fluorescent probes to genetically and/or chemically adhere to their surface. A new class of protein cage nanoparticles, encapsulin, was developed as a tunable dual-functional nanoplatform, which has its target-specific capability with multiple combinations of targeting ligands and colors, using bacterial glue, the SpyTag/SpyCatcher (ST/SC) protein ligation system.

Next, target-specific signal amplifiers were developed as secondary antibody mimics to be applied in immunoassays. Immunoassays are utilized to selectively detect and quantify lowabundance biomolecules in biological samples through antigen-antibody interaction. HRPconjugated IgG-binding nanobodies were established using ST/SC protein ligation system. They have selective and strong binding to specific IgG and show signal amplifying capability in various types of immunoassays, such as western blot, ELISA, and the multiplex TSA cell and tissue imaging.

A variety of target-specific labeling probes demonstrated that they could be utilized in fluorescent cell imaging and immunoassays by combining fluorescent molecules or signal generating enzyme with targeting ligands, including affibody molecules or nanobodies.