Fluorescence energy transfer-based multiplexed hybridization assay using gold nanoparticles and quantum dot conjugates on photonic crystal beads

A multiplexed assay strategy was developed for the detection of nucleic acid hybridization. It is based on fluorescence resonance energy transfer (FRET) between gold nanoparticles (AuNPs) and multi-sized quantum dots (QDs) deposited on the surface of silica photonic crystal beads (SPCBs). The SPCBs were first coated with a three-layer primer film formed by the alternating adsorption of poly(allylamine hydrochloride) and poly(sodium 4-styrensulfonate). Probe DNA sequences were then covalently attached to the carboxy groups at the surface of the QD-coated SPCBs. On addition of DNA-AuNPs and hybridization, the fluorescence of the donor QDs is quenched because of the close proximity of the AuNPs. However, the addition of target DNA causes a recovery of the fluorescence of the QD-coated SPCBs, thus enabling the quantitative assay of hybridized DNA. Compared to fluorescent dyes acting as acceptors, the use of AuNPs results in much higher quenching efficiency. The multiplexed assay displays a wide linear range, high sensitivity, and very little cross-reactivity. This work, where such SPCBs are used for the first time in a FRET assay, is deemed to present a new and viable approach towards high-throughput multiplexed gene assays.