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 identificationof species and species modifications/mutations. Existing technologies (e.g. microarrays, multiplexedbead-based assays, and single use solution assays) are not ideally suited for the demands of many areas ofapplication. It is the combination of cost-effective, repetitive, fast, selective and quantitative response for lowquantities of nucleic acid sequences that is beyond reach of conventional methods, yet which is critical foranalyses in many areas of food safety and quality, clinical diagnostics and biosafety. The goal of the proposedresearch is to develop a quantitative high-throughput nucleic acid diagnostic system that is suitable for rapiddetection of well-defined target nucleic acids. Fluorescent semiconductor quantum dots (QD) will be coatedwith fluorescent nucleic acid probe molecules to create nanoscale biosensors that operate by emitting certaincolours of light when the probe molecules bind to target nucleic acid sequences (hybridization). The QDs willserve as excitation sources to stimulate emission from intrinsically labelled DNA probes when hybridizationoccurs, so that labelling of target nucleic acids will not be necessary. Excitation will be based on FluorescenceResonance Energy Transfer (FRET) from the QDs to the labels that are tethered to the probe molecules. QDs ofdifferent size offer different narrow band emission wavelengths using only one excitation wavelength. Uniquelabelled DNA probe sequences will be associated with differently sized QDs to prepare distinctive QDbiosensors. Mixtures of such QD biosensors have multi-colour emission and a number of target nucleic acidscan be simultaneously determined. Ensembles of such mixtures of different QD biosensors will beimmobilized within microfluidics channels. Electroosmotically driven fluid flow will be used to achievedynamic manipulation of stringency to control selectivity and regeneration of the QD biosensors. The intentionis to create a reversible high throughput sensing system that is suitable for concurrent analysis of multiplenucleic acid targets without the need for labelling of the targets.