Towards Solid-phase Nucleic Acid Hybridization Assays on a Paper-based Platform and Within Channel Microfluidics Using Quantum Dots as Donors in Fluorescence Resonance Energy Transfer

The work described herein has explored solid-phase transduction of nucleic acid hybridization on platforms composed of paper substrates, and also glass surfaces within glass/polydimethylsiloxane microfluidic channels. Single-stranded probe oligonucleotides were conjugated to quantum dots (QDs), and the QDs were immobilized to the solid substrates. The QDs served as donors in fluorescence resonance energy transfer (FRET). Hybridization of solution-phase targets with the probes brought acceptor dye in close proximity to the surface of immobilized QDs, triggering a FRET-sensitized emission from the acceptor dye for ratiometric transduction of nucleic acid hybridization. Chemical modifications of the paper and glass surfaces were developed to immobilize the QD-probe conjugates. Assays with both the paper and glass platforms exhibited fast hybridization kinetics with complete signal development within minutes. For the channel microfluidic experiments, the quantification of target was determined from the spatial length of the microfluidic channel that provided an optical emission indicative of the presence of hybrids. For the paper-based QD-FRET assays, quantification was based on the magnitude of donor and acceptor photoluminescence intensities. The intensity of emission from the paper-based assays was determined using an epifluorescence microscope, and also a consumer digital camera. A novel signal enhancement strategy for QD-FRET transduction was demonstrated on paper substrates. Drying compacted the paper and increased the optical cross-talk of QD-FRET probes to enhance emission intensity. Multiplexed detection of two oligonucleotide targets with excellent mismatch discrimination was demonstrated by implementation of two different QD-dye FRET pairs. Both assay platforms offered a detection limit in the femtomole range, and additional isothermal target amplification allowed target detection in the zeptomole range. The work in this thesis demonstrates that integration of the QD-FRET transduction method with microscale fluidic platforms provides for analytical figures of merit that are sufficient to warrant development of field portable and point-of-care nucleic acid diagnostic applications.Ph.D