Abstract
An ex situ nanoparticle DNA detection assay utilizing DNA-modified nanoparticles attached to DNA monolayer gratings on glass substrates is developed. The assay utilizes the simultaneous hybridization of a single stranded DNA (ssDNA) target molecule to both an amine-modified DNA oligonucleotide attached to an amine-reactive glass surface and a thiol-modified DNA oligonucleotide attached to a 13 nm gold nanoparticle. Surface plasmon resonance imaging measurements are used to characterize the two sequential hybridization adsorption processes employed in the assay, and fluorescence microscopy is used to characterize the formation of DNA monolayer gratings via the photopatterning of the amine-reactive glass slides. First order diffraction measurements utilizing incoherent collimated white light source and a 10 nm bandpass filter centered at 600 nm provided quantitative measurements of target ssDNA down to a concentration of 10 pM. Fourth order diffraction measurements employing a HeNe laser and avalanche photodiode were used to detect target ssDNA adsorption from 10 microl of a solution with a concentration as low as 10 fM, corresponding to 60,000 target DNA molecules. This simple yet sensitive grating-based nanoparticle DNA detection assay should be directly applicable for genetic screening, mRNA expression assays, and microRNA profiling.