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

核酸序列可用于细菌和病毒的检测和鉴定，以及物种和物种修饰/突变的鉴定。现有技术（如微阵列、基于多路磁头的分析和一次性溶液分析）并不理想地适合许多应用领域的需求。它结合了成本效益，重复性，快速，选择性和定量的低数量核酸序列，这是传统方法无法达到的，但它对食品安全和质量，临床诊断和生物安全的许多领域的分析至关重要。提出的研究目标是开发一种定量的高通量核酸诊断系统，适用于快速检测定义明确的靶核酸。荧光半导体量子点（QD）将被涂上荧光核酸探针分子，以创建纳米级生物传感器，当探针分子结合到目标核酸序列（杂交）时，通过发射特定颜色的光来工作。当杂交发生时，量子点将作为激发源来刺激内在标记的DNA探针的发射，因此靶核酸的标记将不需要。激发将基于从量子点到连接到探针分子的标记的荧光共振能量转移（FRET）。不同尺寸的量子点仅使用一个激发波长就能提供不同的窄带发射波长。独特标记的DNA探针序列将与不同大小的量子点相关联，以制备独特的量子点生物传感器。这种QD生物传感器的混合物具有多色发射，并且可以同时确定多个目标核酸。这些不同量子点生物传感器的混合物将被固定在微流体通道内。利用电渗透驱动的流体流动来实现严格度的动态操纵，以控制量子点生物传感器的选择性和再生。目的是创建一个可逆的高通量传感系统，适用于多核酸靶标的并发分析，而不需要标记靶标。

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.