Optimized Immobilization of Biomolecules on Nonspherical Gold Nanostructures for Efficient Localized Surface Plasmon Resonance Biosensing

Plasmonic biosensing techniques employ metal nanostructures, commonly gold (Au), often with biomolecules attached to their surfaces either directly or via other linkers. Various surface chemistry methods based on dispersion and covalent interactions are used to attach biomolecules to Au. As a result, when immobilizing a molecule on a metal surface, quantitative estimates of binding efficiency and stability of these surface chemistry methods are needed. Most prior work to compare such methods deals with bulk/thin film configurations or spherical nanoparticles, and very little is known about immobilization of biomolecules on plasmonic nanostructures of different shapes. Besides, due to rapid advancement of modern nanofabrication techniques, there is a growing need to determine an efficient surface chemistry method for immobilization of biomolecules on nonspherical plasmonic nanostructures. Previous comparison of immobilization methods on spherical Au nanoparticles has shown that physical adsorption resulted in the highest concentration of immobilized antibodies. In our work, we conducted a similar study and compared four representative Au surface functionalization methods as well as estimated how efficient these methods are at attaching biomolecules to nonspherical plasmonic Au nanostructures. We estimated the concentration of immobilized antibody that is specific to human C-reactive protein (anti-hCRP) by measuring the localized surface plasmon resonance (LSPR) shifts after exposing the surface of Au nanostructures to the antibody. Our results differ from the previously reported ones since the highest concentration of anti-hCRP was immobilized using 11-mercaptoundecanoic acid (MUA) chemistry. We demonstrated that immobilized antibodies retained their stability and specificity toward hCRP throughout the immunoassay when diluted hCRP or hCRP-spiked human serum samples were used. These findings have important implications for the fields of biosensing and diagnostics that employ nonspherical plasmonic nanostructures since an overall performance of these devices depends on efficient biomolecule immobilization.