Asymmetrically Functionalized Antibody–Gold Nanoparticle Conjugates to Form Stable Antigen-Assembled Dimers

Abstract

Biomolecular assays based on the aggregation of modified gold nanoparticles (AuNPs) have been developed to provide low detection limits and rapid results with a simple one-step, wash-free procedure. However, a relatively narrow dynamic range, low sensitivity, and poor precision due to time-sensitive readout limit the application of these assay platforms. In this work we synthesized asymmetrically functionalized antibody-AuNP conjugates that are rationally designed to overcome the limitations of aggregation-based immunoassays. Solid-phase synthesis was used to chemically passivate the majority of the AuNP surface and restrict antibody immobilization into a small area of the AuNP surface. These asymmetric conjugates assembled into dimers with the addition of antigen and were stable for over 24 h. In contrast, conventional antibody-AuNP conjugates which are symmetrically modified with antibody assembled into large aggregates that continuously increased in size with the addition of target antigen. These results suggest that asymmetric antibody-AuNP conjugates have the potential to significantly improve the analytical performance of aggregation-based immunoassays.

Effect of the Protein Corona on Antibody–Antigen Binding in Nanoparticle Sandwich Immunoassays

“…We also explored the use of a noncovalent attachment method where Abs were added to the NPs at an incubation ratio of 100:3, and the Abs were allowed to adsorb onto the surface of the NPs (Supporting Information, Figure S4). 25–27 …”

A Plasmonic Approach to Study Protein Interaction Kinetics through the Dimerization of Functionalized Ag Nanoparticles

“…These asymmetric conjugates assembled into dimers with the addition of antigen. The present methodology applied to this dimerization process should be able to elucidate the relevant kinetics information for the design of an immunoassay able to quantify antigens at very small concentrations, improving the performance of other aggregation-based immunoassays 40–44 .…”

Recent Advances in the Generation of Antibody–Nanomaterial Conjugates

“…Importantly, this system has the potential to improve analytical performance of nanoparticleenabled aggregation assays as detection methods with respect to precision, sensitivity, and stability. [72] One particular challenge that has thwarted the mainstream application of physical phenomena for the assembly of nanomaterial-antibody conjugates is the inability to attach GNPs to different types of proteins in a reproducible manner, while producing conjugates that have optical properties akin to each other. This is important for protein-gold conjugates that are used in detection applications, as well as in therapeutics.…”