Toward a multiplexed solid-phase nucleic acid hybridization assay using quantum dots as donors in fluorescence resonance energy transfer.
Solid-phase assays using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET) have been developed for the selective detection of nucleic acids. QDs were immobilized on optical fibers and conjugated with probe oligonucleotides. Hybridization with acceptor labeled target oligonucleotides generated FRET-sensitized acceptor fluorescence that was used as the analytical signal. A sandwich assay was also introduced and avoided the need for target labeling. Green and red emitting CdSe/ZnS QDs were used as donors with Cy3 and Alexa Fluor 647 acceptors, respectively. Quantitative measurements were made via spectrofluorimetry or fluorescence microscopy. Detection limits as low as 1 nM were obtained, and the discrimination of single nucleotide polymorphisms (SNPs) with contrast ratios as high as 31:1 was possible. The assays retained their selectivity and at least 50% of their signal when tested in bovine serum and against a large background of noncomplementary genomic DNA. Mixed films of the two colors of QD and two probe oligonucleotide sequences were prepared for multiplexed solid-phase hybridization assays. It was possible to simultaneously detect two target sequences with retention of selectivity, including SNP discrimination. This research provides an important precedent and framework for the future development of QD-based bioassays and biosensors.
Algar
W Russ
WR
Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, ON L5L 1C6, Canada.
Krull
Ulrich J
UJ
D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D045663:Quantum Dots; D013050:Spectrometry, Fluorescence