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

核酸序列可用于检测和鉴定细菌和病毒，以及鉴定物种和物种修饰/突变。现有技术（例如微阵列、基于多路复用珠的测定和单次使用溶液测定）并不理想地适合于许多应用领域的需求。它是对低数量核酸序列的成本效益、重复性、快速、选择性和定量反应的组合，这是常规方法无法达到的，但对于食品安全和质量、临床诊断和生物安全的许多领域的分析至关重要。本研究的目的是开发一种适用于快速检测明确的靶核酸的定量高通量核酸诊断系统。荧光半导体量子点（QD）将被荧光核酸探针分子涂覆以创建纳米级生物传感器，该纳米级生物传感器通过在探针分子与靶核酸序列结合（杂交）时发射某些颜色的光来操作。当杂交发生时，QD将用作激发源以刺激来自固有标记的DNA探针的发射，使得靶核酸的标记将不是必需的。激发将基于从QD到拴系到探针分子的标记的荧光共振能量转移（FRET）。不同尺寸的QD仅使用一个激发波长提供不同的窄带发射波长。独特的标记的DNA探针序列将与不同大小的QD相关联，以制备独特的QD生物传感器。这种QD生物传感器的混合物具有多色发射，并且可以同时测定许多靶核酸。 不同QD生物传感器的这种混合物的集合将被固定在微流体通道内。电驱动的流体流动将用于实现严格性的动态操纵，以控制QD生物传感器的选择性和再生。本发明的目的是创造一种可逆的高通量传感系统，其适合于同时分析多个核酸靶，而不需要标记靶。

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.