Preparation of paramagnetic microparticles and their applications in the study of ligand -receptor binding characteristics
Abstract
Single molecule force measurements have made it possible to directly characterize inter- and intramolecular interactions between biological macromolecules and their assemblies, respectively. In this program of research, a new magnetic tweezers technique was developed for the measurement of the bond lifetime of the immunoglobulin G (IgG)-protein A Interaction. This has been achieved through the development of paramagnetic microparticles with high and uniform magnetization and the design of a permanent magnet that is capable of creating high magnetic fields and field gradients over macroscopic areas. The paramagnetic microparticles were created with the emulsion templated technique. These microparticles were synthesized using four steps: creation of an oil-in-water emulsion in which hydrophobic iron oxide nanoparticles and UV activated initiator were distributed in hexane; formation of uniform microparticles through emulsion homogenization and evaporation of hexane; functionalization of the microparticle with a PEG functionalized surfactant and acrylic acid; and polymerization of the microparticles. The size, iron content, polymer content, magnetic properties, and surface chemistry of these particles were characterized using transmission electron microscopy (TEM), thermogravimetric analysis (TGA), atomic adsorption spectroscopy (AA), superconducting quantum interference device (SQUID) magnetometry, x-ray photoelectron spectroscopy (XPS), and colloidal stability analysis. The resulting microparticles had a size, polymer content, magnetic properties, and surface chemistry that were ideally suited for biophysical measurements and bioanalytical techniques. The detachment of hundreds of superparamagnetic microparticles from a surface at a constant force was monitored optically, and the resulting particle behavior provided a direct measure of the form of the IgG-protein A interactions between the microparticle and surface. The off-rate and effective bond length of the specific molecular interaction were measured at protein densities where single molecule interactions occurred. The fact that these measurements were simultaneously made between a large populations of ligand-receptor pairs promises to allow molecular forces to be manipulated in diagnostic assays and high-throughput screening of ligand-receptor interactions.
Degree
Ph.D.
Advisors
Lee, Purdue University.
Subject Area
Biomedical engineering|Chemical engineering
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