FRET-Based Solid-Phase Three-Color and Four-Color Hybridization Assays Using Mixed Films of Quantum Dots and Oligonucleotides

摘要

Mixed films of CdSe/ZnS quantum dots (QDs) and oligonucleotide probes were immobilized on optical fibers for the multiplexed detection of nucleic acids using fluorescence resonance energy transfer (FRET). Green and red emitting QDs were energy donors for three different acceptor dyes: Cy3, Rhodamine Red-X, and Alexa Fluor 647. The simultaneous detection of three different nucleic acid targets (three-plex) was possible using a sandwich format, where probe/target/reporter hybridization provided the proximity for FRET. This approach enabled multiplexing at the ensemble level on a single substrate, and without spatial registration of probes or multiple excitation sources. Through the combination of these FRET pairs and the direct excitation of a fourth dye, Pacific Blue, it was possible to simultaneously detect four different nucleic acid targets (four-plex). The main limitation of this approach was low dye fluorescence signals caused by probe dilution in the mixed film. The use of reporter oligonucleotides labeled with multiple acceptor dye molecules was proposed as a route to signal enhancement. Proof-of-concept experiments with dual label reporter oligonucleotides confirmed this hypothesis. Further development of signal enhancement strategies may lead to a QD and FRET-based four-plex assay suitable for bioanalytical and biomedical applications.