Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, ON L5L 1C6, Canada.
Anal Bioanal Chem. 2011 Jan;399(1):133-41. doi: 10.1007/s00216-010-4309-0. Epub 2010 Oct 27.
The optical properties and surface area of quantum dots (QDs) have made them an attractive platform for the development of nucleic acid biosensors based on fluorescence resonance energy transfer (FRET). Solid-phase assays based on FRET using mixtures of immobilized QD-oligonucleotide conjugates (QD biosensors) have been developed. The typical challenges associated with solid-phase detection strategies include non-specific adsorption, slow kinetics of hybridization, and sample manipulation. The new work herein has considered the immobilization of QD biosensors onto the surfaces of microfluidic channels in order to address these challenges. Microfluidic flow can be used to dynamically control stringency by adjustment of the potential in an electrokinetic-based microfluidics environment. The shearing force, Joule heating, and the competition between electroosmotic and electrophoretic mobilities allow the optimization of hybridization conditions, convective delivery of target to the channel surface to speed hybridization, amelioration of adsorption, and regeneration of the sensing surface. Microfluidic flow can also be used to deliver (for immobilization) and remove QD biosensors. QDs that were conjugated with two different oligonucleotide sequences were used to demonstrate feasibility. One oligonucleotide sequence on the QD was available as a linker for immobilization via hybridization with complementary oligonucleotides located on a glass surface within a microfluidic channel. A second oligonucleotide sequence on the QD served as a probe to transduce hybridization with target nucleic acid in a sample solution. A Cy3 label on the target was excited by FRET using green-emitting CdSe/ZnS QD donors and provided an analytical signal to explore this detection strategy. The immobilized QDs could be removed under denaturing conditions by disrupting the duplex that was used as the surface linker and thus allowed a new layer of QD biosensors to be re-coated within the channel for re-use of the microfluidic chip.
量子点 (QD) 的光学性质和表面积使其成为基于荧光共振能量转移 (FRET) 的核酸生物传感器发展的理想平台。已经开发了基于 FRET 的固相测定法，该方法使用固定化 QD-寡核苷酸缀合物的混合物 (QD 生物传感器)。与固相检测策略相关的典型挑战包括非特异性吸附、杂交动力学缓慢以及样品处理。本文的新工作考虑了将 QD 生物传感器固定在微流道的表面上，以解决这些挑战。可以使用微流体流通过在基于电动的微流体环境中调整电势来动态控制严格性。剪切力、焦耳加热以及电渗流和电泳迁移率之间的竞争允许优化杂交条件、将目标物以对流方式输送到通道表面以加速杂交、改善吸附以及再生传感表面。微流体流还可用于输送 (用于固定化) 和去除 QD 生物传感器。使用与两种不同寡核苷酸序列缀合的 QD 来证明其可行性。QD 上的一个寡核苷酸序列可用作通过与位于微流道内玻璃表面上的互补寡核苷酸杂交而进行固定化的接头。QD 上的第二个寡核苷酸序列用作探针，以在样品溶液中的靶核酸上进行杂交。靶上的 Cy3 标记物通过使用绿色发射的 CdSe/ZnS QD 供体进行 FRET 激发，并提供分析信号以探索这种检测策略。通过破坏用作表面接头的双链体，可以在变性条件下去除固定化的 QD，从而允许在通道内重新涂覆新的 QD 生物传感器层，以重新使用微流控芯片。
