Formation, Characterization, and Optimization of Antibody-Gold Nanoparticle Conjugates

Protein modified gold nanoparticle based immunoassays are the basis of many novel detection techniques. There are many groups working on novel immunoassays but there is still much to understand about how many proteins are attached onto each nanoparticle and much to improve on immobilization methods. This thesis work is devoted to improving techniques for the quantification of protein immobilized on the gold nanoparticle and development of a novel approach to immobilize protein independent of pH. The ability to evaluate antibody immobilization onto gold nanoparticles is critical for assessing coupling chemistry and optimizing the sensitivity of nanoparticle-enabled biosensors. Herein, we developed a fluorescence-based method for directly quantifying antibodies bound onto gold nanoparticles. Antibody-modified gold nanoparticles were treated with KI/I2 etchant to dissolve the gold nanoparticles. A desalting spin column was used to recover the antibody released from the nanoparticles, and NanoOrange, a fluorescent dye, was used to quantify the antibody. We determined 309 ± 93 antibodies adsorb onto each 60 nm gold nanoparticle (2.6 × 1010 NP/mL), which is consistent with a fully adsorbed monolayer based on the footprint of an IgG molecule. Moreover, the increase in hydrodynamic diameter of the conjugated nanoparticle (76 nm) compared to that of the unconjugated nanoparticle (62 nm) confirmed that multilayers did not form. A more conventional method of indirectly quantifying the adsorbed antibody by analysis of the supernatant overestimated the antibody surface coverage (660 ± 87 antibodies per nanoparticle); thus, we propose the method described herein as a more accurate alternative to the conventional approach. The immobilization of antibody onto gold nanoparticles is important for many novel nanoparticle based immunoassays. Current methods of immobilization are limited by the inability to immobilize antibody onto gold nanoparticles over a range of pH values. Direct adsorption requires the pH to be slightly higher than the isoelectric point of the antibody and covalent attachment via bifunctional crosslinking chemistry molecules requires a specific pH as well. This is an issue when working with multiple antibodies at once for multiplex detection. In this thesis, we present a new method of immobilization by which the antibody is modified via a molecule with a N-hydroxysuccinimidyl ester group, then adsorbed onto a gold nanoparticle. We demonstrate that modification of antibodies allows for adsorption onto gold nanoparticles independent of pH. Furthermore, we show that this modification method is applicable to multiple antibodies. Finally, we show that these modified antibodies are active and comparable to conventional assays