Agglomerates of Gold Nanoparticles Based on the Biotin–Streptavidin System for Lateral Flow Immunoassay

Abstract

Spherical GNPs of various sizes (10–30 nm), obtained by the reduction of chloroauric acid with ascorbic acid, are used to obtain conjugates with native and biotinylated antibodies (Ab, bAb). The process of the formation of nanoagglomerates of GNP-bAb conjugates with the participation of free streptavidin (Stvd) in a solution is studied in the range of stoichiometric ratios [Stvd] : [GNP] from 0 : 1 to 3200 : 1. Using the dynamic-light-scattering method, it is shown that at a 19–50-fold excess of free Stvd relative to GNPs in a solution, GNP-bAb conjugates form nanoagglomerates with an effective diameter 1.5–3 times larger than the size of the initial conjugate. The efficiency of the interaction of a detection reagent prepared from a GNP-bAb conjugate in the presence of various concentrations of free Stvd in a lateral flow with Stvd or bAb absorbed on a membrane is studied. It is shown that the color intensity of the reaction zones undergoes a significant change in the region of the ratio of the concentrations of GNP-bAb and Stvd, corresponding to the formation of nanoagglomerates, and depends on the composition and size of the resulting complex and the presence in its composition of Stvd molecules or biotin residues available for binding with the absorbed component. On the basis of mathematical modeling of the proposed kinetic mechanism for the formation of GNP complexes with the participation of biotin-streptavidin interaction, the predominant formation of nanoagglomerates consisting of two or three GNPs linked by Stvd molecules is shown. The use of such nanoagglomerates as a label in a lateral immunoassay can lead to a several-fold reduction in the detection limit of the assay.