A real-time PCR-based method for determining the surface coverage of thiol-capped oligonucleotides bound onto gold nanoparticles.
Here we report a real-time PCR-based method for determining the surface coverage of dithiol-capped oligonucleotides bound onto gold nanoparticles alone and in tandem with antibody. The detection of gold nanoparticle-bound DNA is accomplished by targeting the oligonucleotide with primer and probe binding sites, amplification of the oligonucleotide by PCR, and real-time measurement of the fluorescence emitted during the reaction. This method offers a wide dynamic range and is not dependant on the dissociation of the oligonucleotide strands from the gold nanoparticle surface; the fluorophore is not highly quenched by the gold nanoparticles in solution during fluorescence measurements. We show that this method and a fluorescence-based method give equivalent results for determining the surface coverage of oligonucleotides bound onto 13 or 30 nm gold nanoparticles alone and in tandem with antibody. Quantifying the surface coverage of immobilized oligonucleotides on metallic nanoparticle surfaces is important for optimizing the sensitivity of gold nanoparticle-based detection methods and for better understanding the interactions between thiol-functionalized oligonucleotides and gold nanoparticles.
Kim
Eun-Young
EY
Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Stanton
Jennifer
J
Vega
Rafael A
RA
Kunstman
Kevin J
KJ
Mirkin
Chad A
CA
Wolinsky
Steven M
SM
D004247:DNA / Q000032:analysis*; D005456:Fluorescent Dyes / Q000737:chemistry; D006046:Gold / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D015345:Oligonucleotide Probes / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature; D013873:Thionucleotides / Q000737:chemistry*; D013997:Time Factors
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D013873:Thionucleotides; D006046:Gold; D004247:DNA