Engineering DNA Nanocube SAM Scaffolds for FRET-Based Biosensing: Interfacial Characterization and Sensor Demonstration

Decorating a gold surface with molecular-level control over the positioning of DNA probes was demonstrated using a self-assembled monolayer (SAM) of wireframe DNA nanocube structures. The DNA nanocubes were specifically adsorbed and oriented using thiol-modified DNA on one face of the cube. The DNA nanocube SAM had a uniform coverage over the gold single crystal bead electrode with a separation of 20-30 nm measured by AFM. The face of the nanocube furthest from the gold surface was designed to hybridize with two different sequences of a 50 base single-stranded DNA probe that was modified with a fluorophore. The first 20 bases were hybridized with the DNA nanocube. One of a pair of FRET fluorophores was used for each probe strand. The dimensions of the nanocube controlled the relative spacing between these fluorophores. When the DNA probes were single-stranded, a FRET signal was observed. FRET decreased to background levels when a complementary DNA target was hybridized to either probe, resulting in a turn-off sensor with little cross-talk between the individual hybridization events. Hybridization isotherms for one target gave