Localized surface plasmon resonance based gold nanoparticle-hairpin assay for optical detection of single DNA molecules

Without labeling-steps, localized surface plasmon resonance (LSPR) lacks sensitivity towards small molecules, like DNA. Available labeling methods experience coupling-distance limitations and/or consist of multiple assay steps. In this research an optical single sensing-step DNA sensor using hairpin-DNA without limiting target-DNA lengths was developed. The sensor read-out is based on the displacement of gold nanoparticles (AuNPs) attached to hairpin-DNA relative to an Au-surface on target binding. The relative distance between AuNP and Au-surface increases upon hybridization of hairpin-DNA with target-DNA. Binding of individual target DNA strands can be observed by darkfield microscopy as a color change from red to green due to distance-limited coupling of AuNPs to Au-surfaces.

Hairpin-opening in the presence of target-DNA was confirmed by Förster Resonance Energy Transfer and gel electrophoresis. The sensor consists of HS-hairpin-NH2 DNA immobilized on thiol alkane ethylene-glycol self-assembled monolayers via EDC/NHS coupling. AuNPs are immobilized through thiol chemistry. Assembly steps and sequence specificity of the sensor are confirmed by Quartz Crystal Microbalance (QCM) measurements.

Darkfield microscopy confirms the AuNP displacement in the presence of target-DNA, which is clearly visible as a colour change from red to green (Figure 2). Future research will focus on analysing/observing colour shifts of multiple gold nanoparticle-hairpin structures simultaneously, and is expected to allow optical detection of exceptionally low DNA concentrations.