ABSTRACT: DNA-functionalized gold nanoparticles have been increasingly applied as sensitive and selective analytical probes
and biosensors. The DNA ligands bound to a nanoparticle dictate its reactivity, making it essential to know the type and number of
DNA strands bound to the nanoparticle surface. Existing methods used to determine the number of DNA strands per gold nanoparticle
(AuNP) require that the sequences be fluorophore labeled, which may affect the DNA surface coverage and reactivity of the
nanoparticle, and/or require specialized equipment and other fluorophore-containing reagents. We report a UV-visible based method
to conveniently and inexpensively determine the number of DNA strands attached to AuNPs of different core sizes. When this
method is used in tandem with a fluorescence dye assay, it is possible to determine the ratio of two unlabeled sequences of different
lengths bound to AuNPs. Two sizes of citrate-stabilized AuNPs (5nm and 12 nm) were functionalized with mixtures of short (5
base) and long (32 base) disulfide-terminated DNA sequences and the ratios of sequences bound to the AuNPs were determined
using the new method. The long DNA sequence was present as a lower proportion of the ligand shell than in the ligand exchange
mixture, suggesting it had a lower propensity to bind the AuNPs than the short DNA sequence. The ratio of DNA sequences bound
to the AuNPs was not the same for the large and small AuNPs, which suggests that the radius of curvature had a significant influence
on the assembly of DNA strands onto the AuNPs.