Solid-Phase Resonance Energy Transfer Based Bioassays Using Immobilized Upconverting Nanoparticles as Donors and Quantum Dots as Acceptors

The work herein describes the development of interfacial bioassays for the detection of biomolecules on glass and paper substrates. Monodisperse water soluble upconverting nanoparticles (UCNPs) were covalently immobilized on modified cover slips and cellulose paper for the development of bioassays. Investigations included examination of the effectiveness of luminescence resonance energy transfer (LRET) for optical signal generation. This work presents the first account of covalent immobilization of UCNPs for use as energy donors. The functionality and stability of the immobilized UCNPs as donors was demonstrated by use of quantum dots (QDs) as energy acceptors in configurations suitable for LRET. A monolayer of densely packed UCNPs was achieved on glass cover slips and was used for the detection of thrombin using selective aptamers. On paper, a sandwich assay for the detection of unlabeled oligonucleotide targets offered a limit of detection in the femtomole range and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs provided improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was enhanced by the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The assay was extended to the simultaneous detection of three targets using a single form of UCNP as donor and three different color emitting QDs as acceptors. The intensity of emission from each nanoparticle was determined using an epifluorescence microscope and an optical band-pass filter without the need for spectral deconvolution. An instrument with luminescence lifetime measurement capabilities was assembled to investigate the efficiency of resonance energy transfer. The work presented in this thesis describes the essential elements required to assemble a LRET-based assay for the sensitive and selective multiplexed detection of nucleic acids on paper using UCNPs as energy donors.Ph.D