High throughput nucleic acid detection systems based on multiplexing using quantum dots

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Nucleic acid sequences can be used for detection and identification of bacteria and viruses, and identification of species and species modifications/mutations. Existing technologies (e.g. microarrays, multiplexed bead-based assays, and single use solution assays) are not ideally suited for the demands of many areas of application. It is the combination of cost-effective, repetitive, fast, selective and quantitative response for low quantities of nucleic acid sequences that is beyond reach of conventional methods, yet which is critical for analyses in many areas of food safety and quality, clinical diagnostics and biosafety. The goal of the proposed research is to develop a quantitative high-throughput nucleic acid diagnostic system that is suitable for rapid detection of well-defined target nucleic acids. Fluorescent semiconductor quantum dots (QD) will be coated with fluorescent nucleic acid probe molecules to create nanoscale biosensors that operate by emitting certain colours of light when the probe molecules bind to target nucleic acid sequences (hybridization). The QDs will serve as excitation sources to stimulate emission from intrinsically labelled DNA probes when hybridization occurs, so that labelling of target nucleic acids will not be necessary. Excitation will be based on Fluorescence Resonance Energy Transfer (FRET) from the QDs to the labels that are tethered to the probe molecules. QDs of different size offer different narrow band emission wavelengths using only one excitation wavelength. Unique labelled DNA probe sequences will be associated with differently sized QDs to prepare distinctive QD biosensors. Mixtures of such QD biosensors have multi-colour emission and a number of target nucleic acids can be simultaneously determined.  Ensembles of such mixtures of different QD biosensors will be immobilized within microfluidics channels. Electroosmotically driven fluid flow will be used to achieve dynamic manipulation of stringency to control selectivity and regeneration of the QD biosensors. The intention is to create a reversible high throughput sensing system that is suitable for concurrent analysis of multiple nucleic acid targets without the need for labelling of the targets.