A Fluorescence-Based Method for Determining the Surface Coverage and Hybridization Efficiency of Thiol-Capped Oligonucleotides Bound to Gold Thin Films and Nanoparticles

TLDR

A fluorescence‑based assay was used to quantify thiol‑capped oligonucleotide binding to gold nanoparticles and to tailor surface coverage via co‑adsorbed diluent strands. The study found that gold nanoparticles exhibit higher oligonucleotide surface coverage but lower hybridizable strand density than thin films, and that increasing electrolyte concentration and adding spacer sequences markedly enhance coverage, stability, and hybridization efficiency, enabling better control of hybridized strands per particle and improving nanoparticle‑based detection.

Abstract

Using a fluorescence-based method, we have determined the number of thiol-derivatized single-stranded oligonucleotides bound to gold nanoparticles and their extent of hybridization with complementary oligonucleotides in solution. Oligonucleotide surface coverages of hexanethiol 12-mer oligonucleotides on gold nanoparticles (34 ± 1 pmol/cm2) were significantly higher than on planar gold thin films (18 ± 3 pmol/cm2), while the percentage of hybridizable strands on the gold nanoparticles (1.3 ± 0.3 pmol/cm2, 4%) was lower than for gold thin films (6 ± 2 pmol/cm2, 33%). A gradual increase in electrolyte concentration over the course of oligonucleotide deposition significantly increases surface coverage and consequently particle stability. In addition, oligonucleotide spacer sequences improve the hybridization efficiency of oligonucleotide-modified nanoparticles from ∼4 to 44%. The surface coverage of recognition strands can be tailored using coadsorbed diluent oligonucleotides. This provides a means of indirectly controlling the average number of hybridized strands per nanoparticle. The work presented here has important implications with regard to understanding interactions between modified oligonucleotides and metal nanoparticles, as well as optimizing the sensitivity of gold nanoparticle-based oligonucleotide detection methods.