Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, North, Mississauga, Ontario, L5L 1C6, Canada.
Langmuir. 2010 Apr 20;26(8):6041-7. doi: 10.1021/la903751m.
Methods have been developed for the simultaneous and selective detection of three target nucleic acid sequences based on mixed films of immobilized quantum dots (QDs) and oligonucleotide probes. CdSe/ZnS QDs were immobilized on optical fibers and conjugated with mixtures of different probe oligonucleotides. Hybridization events were detected using a combination of fluorescence from direct excitation and fluorescence sensitized by resonance energy transfer (FRET). A sandwich assay format was used to associate dye labeled reporter oligonucleotides with probe-target hybrids formed at the surface of the optical fiber. One detection channel utilized direct excitation of Pacific Blue and the two other detection channels were based on FRET. In one strategy, green emitting QDs were used as donors with Cy3 and Rhodamine Red-X acceptors. In a second strategy, green and red emitting QDs were coimmobilized and used as donors with Cy3 and Alexa Fluor 647 acceptors, respectively. Selective three-plex detection was demonstrated with both strategies. Several key design criteria that were explored to optimize the relative signal magnitude between channels included: the ratio of probe associated with direct excitation versus probes associated with FRET; the relative amounts of each FRET probe and corresponding spectral overlap; and the photoluminescence ratio between immobilized green and red emitting QDs (where applicable). Careful selection of probe sequences and lengths were important for the discrimination of single nucleotide polymorphisms in one channel without suppressing binding of target in the other two channels. This work provides a basis for the development of multiplexed biosensors that are ensemble compatible and do not require discrete sensor elements, spatial registration, sorting technology, or single molecule spectroscopy.
已经开发出了基于固定化量子点（QD）和寡核苷酸探针混合膜的同时和选择性检测三种靶核酸序列的方法。CdSe/ZnS QD 被固定在光纤上，并与不同探针寡核苷酸的混合物偶联。杂交事件通过直接激发的荧光和共振能量转移（FRET）敏化的荧光的组合来检测。夹心测定法用于将染料标记的报告寡核苷酸与在光纤表面形成的探针-靶杂交体结合。一个检测通道利用 Pacific Blue 的直接激发，另外两个检测通道基于 FRET。在一种策略中，绿色发射 QD 用作供体，Cy3 和 Rhodamine Red-X 作为受体。在第二种策略中，将绿色和红色发射 QD 共固定，并分别将 Cy3 和 Alexa Fluor 647 用作供体和受体。两种策略都实现了选择性的三重检测。为了优化通道之间的相对信号幅度，探索了几个关键的设计标准，包括：与直接激发相关的探针与与 FRET 相关的探针的比例；每种 FRET 探针的相对量和相应的光谱重叠；以及固定化绿色和红色发射 QD 之间的光致发光比（在适用的情况下）。仔细选择探针序列和长度对于在一个通道中区分单核苷酸多态性而不抑制其他两个通道中靶的结合非常重要。这项工作为开发不需要离散传感器元件、空间配准技术、分类技术或单分子光谱学的集总兼容的多路复用生物传感器提供了基础。
