Toward an On-chip Multiplexed Nucleic Acid Hybridization Assay Using Immobilized Quantum Dot-Oligonucleotide Conjugates and Fluorescence Resonance Energy Transfer

Semiconductor quantum dots (QD) are a class of NP with photophysical properties that are ideally suited for optical multiplexing and use as donors in fluorescence resonance energy transfer (FRET). A new strategy is presented for the development of multiplexed DNA hybridization assays using immobilized QDs in a microfluidic system. Green-or red-emitting QDs were immobilized via self-assembly with a multidentate-thiol-derivatized glass slide, and subsequently conjugated with amine-terminated probe oligonucleotides using carbodiimide activation. Immobilized QD-probe conjugates were then passivated with adsorbed non-complementary oligonucleotides to achieve selectivity in microfluidic assays. Target nucleic acid sequences hybridized with QD-probe conjugates and were labeled with Cy3 or Alexa Fluor 647 as acceptor dyes for the QD donors, where FRET-sensitized dye emission provided a signal for the detection of picomolar quantities of target. The simultaneous immobilization of green-and red-emitting QDs at different ratios within a microfluidic channel was demonstrated as a step toward multiplexed assays.