量子点(QD)的有利光学特性激发了它们在与成像和生物分析相关的各种应用中的用途。包括检测蛋白酶及其活性。最近的研究表明,量子点的表面化学能够调节蛋白酶的活性,但只是非特定的。这里,我们提出了一种策略,以选择性地加速特定靶蛋白酶的活性多达两个数量级。外位点结合“诱饵”肽来自跨越一系列生物学作用的蛋白质-底物,受体,和抑制剂-并用于增加QD-肽缀合物对凝血酶或因子Xa的亲和力,导致共结合底物的蛋白水解速率增加。与QD表面化学的影响不同,加速度对目标蛋白酶具有特异性,而其他蛋白酶的加速度可忽略不计。这种“诱饵和切割”传感方法的好处包括检测极限提高了一个数量级以上,在非目标蛋白水解的压倒性背景下重新启用目标蛋白酶的检测,和缓解抑制剂的作用。累积结果指向可推广的策略,加速的机制,设计诱饵肽和缀合物的考虑因素,并讨论了扩大这种方法范围的途径。总的来说,这项研究代表了在提高灵敏度和选择性的纳米颗粒酶传感器的合理设计方面迈出的重要一步.
The advantageous optical properties of quantum dots (QDs) motivate their use in a wide variety of applications related to imaging and bioanalysis, including the detection of proteases and their activity. Recent studies have shown that surface chemistry on QDs is able to modulate protease activity, but only nonspecifically. Here, we present a strategy to selectively accelerate the activity of a particular target protease by as much as two orders of magnitude.
Exosite-binding \"bait\" peptides were derived from proteins that span a range of biological roles─substrate, receptor, and inhibitor─and were used to increase the affinity of the QD-peptide conjugates for either thrombin or factor Xa, resulting in increased rates of proteolysis for coconjugated substrates. Unlike effects from QD surface chemistry, the acceleration was specific to the target protease with negligible acceleration of other proteases. Benefits of this \"bait and cleave\" sensing approach included detection limits that improved by more than an order of magnitude, reenabled detection of target protease against an overwhelming background of nontarget proteolysis, and mitigation of the action of inhibitors. The cumulative results point to a generalizable strategy, where the mechanism of acceleration, considerations for the design of bait peptides and conjugates, and routes to expanding the scope of this approach are discussed. Overall, this research represents a major step forward in the rational design of nanoparticle-based enzyme sensors that enhance sensitivity and selectivity.