Date of Completion
Dr. James F. Rusling
Dr. Steven L. Suib
Dr. Douglas H. Adamson
Field of Study
Doctor of Philosophy
Protein binding is used extensively in significant biomedical applications essential to modern research and clinical practice including biomolecule purification, in vivo imaging, cancer diagnostics, drug delivery, and bioanalysis. Recently monoclonal antibodies (MAb) are becoming useful in cancer immunotherapy. This new approach to cancer therapy has already led to an $20 billion/year industry, with projected revenue in excess of $80 billion by 2020. They are essential components for immunoassays and other bioanalytical applications as well. While there are a wide variety of commercially available antibodies, isolation and purification from animal hosts and in vitro cell source makes them quite expensive ($300-2000/mg), and storage stability is an issue. Immunotherapeutic MAbs are the world’s most expensive drugs and require more stringent purity than most Abs. Hence, there is a need for cost-effective, fast, stable, resilient systems to purify and separate the antibodies. Additionally, nanoparticles with selective antibody-like binding sites may offer significant advantages in cost and stability
The overall goal of the thesis is to mimic protein-binding sites on nanoparticles using surface molecular imprinting technology. In one of the projects, synthesis of antibody-like binding sites by molecular imprinting on silica nanoparticles (SiNP) using a combination of four organosilane monomers with amino acid-like side chains providing hydrophobic, hydrophilic and H-bonding interactions with target proteins is achieved. This approach provided artificial antibody (AA) nanoparticles with good selectivity and specificity to binding domains on target protein a relatively low-cost synthesis. We made AAs by polymer grafting onto SiNPs for human serum albumin (HSA) and glucose oxidase (GOx), and tested binding affinity, selectivity and specificity vs. several other proteins using adsorption isotherms and surface plasmon resonance (SPR). The Langmuir-Freundlich adsorption model was used to obtain apparent binding constants (KLF) from binding isotherms of HSA (6.7x104) and GOx (4.7x104) to their respective AAs. These values were 4-300 fold larger compared to a series of non-template proteins. SPR binding studies of AAs with proteins attached to a gold surface confirmed good specificity and revealed faster binding for the target proteins compared to non-target proteins. Similarly, we have prepared HSA-binding SiO2@Fe2O3 nanoparticles (NP) with binding constants for HSA 5-250-fold larger than for non-target proteins. We explored the application of the material in proteomics field. These multiply reusable particles removed HSA from human serum equivalent to commercial Albumin removal kits, but at 10-fold lower cost. Our micro-batch approach for synthesizing binding sites on magnetic nanoparticles will result in novel low-cost materials immediately applicable to protein removal from serum. In another project, we developed reusable magnetic core-shell nanoparticles that selectively bind IgG MAb’s and demonstrate purification of selected IgGs from human serum.
Bhakta, Snehasis, "Surface-Imprinted Nanoparticles for Protein Recognition and Separation" (2017). Doctoral Dissertations. 1519.