Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, United States.
Anal Chem. 2012 Nov 20;84(22):10136-46. doi: 10.1021/ac3028068. Epub 2012 Nov 5.
Semiconductor quantum dots (QDs) are attractive probes for optical sensing and imaging due to their unique photophysical attributes and nanoscale size. In particular, the development of assays and biosensors based on QDs and Förster resonance energy transfer (FRET) continues to be a prominent focus of research. Here, we demonstrate the application of QDs as simultaneous donors and acceptors in a time-gated FRET relay for the multiplexed detection of protease activity. In contrast to the current state-of-the-art, which uses multiple colors of QDs, multiplexing was achieved using only a single color of QD. The other constituents of the FRET relay, a luminescent terbium complex and fluorescent dye, were assembled to QDs via peptides that were selected as substrates for the model proteases trypsin and chymotrypsin. Loss of prompt FRET between the QD and dye signaled the activity of chymotrypsin; loss of time-gated FRET between the terbium and QD signaled the activity of trypsin. We applied the FRET relay in a series of quantitative, real-time kinetic assays of increasing biochemical complexity, including multiplexed sensing, measuring inhibition in a multiplexed format, and tracking the proteolytic activation of an inactive pro-protease to its active form in a coupled, multienzyme system. These capabilities were derived from a ratiometric analysis of the two FRET pathways in the relay and permitted extraction of initial reaction rates, enzyme specificity constants, and apparent inhibition constants. This work adds to the growing body of research on multifunctional nanoparticles and introduces multiplexed sensing as a novel capability for a single nanoparticle vector. Furthermore, the ability to track both enzymes within a coupled biological system using one vector represents a significant advancement for nanoparticle-based biosensing. Prospective applications in biochemical research, applied diagnostics, and drug discovery are discussed.
半导体量子点 (QDs) 由于其独特的光物理属性和纳米级尺寸，成为光学传感和成像的有吸引力的探针。特别是，基于 QDs 和Förster 共振能量转移 (FRET) 的测定法和生物传感器的开发仍然是研究的突出重点。在这里，我们展示了将 QDs 用作时间门控 FRET 继电器中的同时供体和受体，用于蛋白酶活性的多重检测。与当前使用多种颜色的 QDs 的最新技术相比，仅使用一种颜色的 QD 实现了多重检测。FRET 继电器的其他组成部分，即发光铽配合物和荧光染料，通过肽组装到 QD 上，这些肽被选为模型蛋白酶胰蛋白酶和糜蛋白酶的底物。QD 和染料之间的即时 FRET 的损失表明糜蛋白酶的活性；铽和 QD 之间的时间门控 FRET 的损失表明胰蛋白酶的活性。我们在一系列定量、实时动力学测定中应用了 FRET 继电器，这些测定的生化复杂性逐渐增加，包括多重传感、以多重格式测量抑制作用，以及跟踪无活性前蛋白酶的蛋白水解激活到其在耦合、多酶系统中的活性形式。这些功能源自继电器中两个 FRET 途径的比率分析，并允许提取初始反应速率、酶特异性常数和表观抑制常数。这项工作增加了关于多功能纳米粒子的研究，并将多重检测引入到单个纳米粒子载体的新型能力中。此外，使用一个载体在耦合生物系统中跟踪两种酶的能力代表了基于纳米粒子的生物传感的重大进展。讨论了在生化研究、应用诊断和药物发现中的潜在应用。
