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

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 approximately 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.

Peptide antisense nanoparticles

“…To characterize the degree of nanoparticle surface functionalization, the numbers of attached oligonucleotides and peptides per Au NP were determined by fluorescence methods (see Materials and Methods) (46). The results of these studies demonstrate that, although there is not a 1:1 correlation, the solution stoichiometry (oligonucleotides and peptides) can be used to control the surface composition of the nanoparticle (Fig.…”

Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles

“…Again, the attachment of larger biomolecules can be observed by the increase of the total particle size as discussed before. In combination with organic fluorophores conjugated to the molecule of interest, the presence of those molecules on the particle surface can be accessed by the absorption or fluorescence emission (Demers et al 2000;Meiser et al 2004;Sari et al 2004;Hurst et al 2006), or by stepwise photobleaching of the organic dye (Casanova et al 2007). …”

Instantaneous Attachment of an Ultrahigh Density of Nonthiolated DNA to Gold Nanoparticles and Its Applications

“…In the first approach, a thiolated DNA is mixed with AuNPs and a high DNA density can be achieved via a process known as 'salt aging' (Figure 1A), where salt is gradually added over 1-2 days so that an increasing number of DNA strands can be adsorbed to form stable conjugates. [12][13][14] Recent improvements of this strategy including the use of surfactants and sonication have drastically reduced the aging time to a few hours and even very large AuNPs could be functionalized. 14,15 Depending on the final salt concentration and DNA sequence, up to ~130 thiolated DNA can be adsorbed on each 13 nm AuNP.…”