Nanotechnology for Multiplex Detection of Enzymes

描述（由申请人提供）：目前的检测平台基本上是为了测定生物样品中的蛋白质表达水平而设计的，但对于酶，不仅是表达水平，而且催化活性对其在病理过程中的功能作用也很重要。我们最近开发了一种量子点生物发光共振能量转移（QD-BRET）纳米系统。当Renilla luciferase突变体（Luc8）与量子点接近时，Luc8氧化底物coelenterazine所产生的生化能量可以通过BRET传递到量子点上，从而在量子点上产生发光。由于这个QD-BRET过程是距离依赖的，它提供了一个独特的检测平台来测定酶的活性。与现有的生物成像和生物检测平台相比，这种QD-BRET纳米系统具有许多优点。首先，通过消除对外部光输入的需要，QD-BRET避免了与荧光检测相关的问题，如背景荧光、直接受体激发和光漂白。其次，它为检测提供了很高的灵敏度。第三，现有的检测技术受到血红蛋白吸光度的干扰，需要分析血清而不是全血。QD-BRET与全血分析兼容，从而避免了对血清制备的需要。第四，QD-BRET平台适用于多路复用，可以在一次测试中分析多个目标。此外，通过避免外部照明源，这项技术更容易小型化，并发展成为便携式诊断设备。第五，与传统的使用量子点的FRET协议相比，QD- bret使用量子点作为发射器，利用其突出的吸收截面和广泛的吸收光谱。本R21申请旨在通过开发具有竞争力的QD-BRET （cQD-BRET）传感器来进一步推进QD-BRET技术，用于生物样品中酶靶点的高灵敏度多重检测，有三个具体目的：1)建立cQD-BRET传感器设计，用于血清、全血和组织样品的均质分析中蛋白酶的多重检测。2)固定化抗体在微孔板读取平台上多重检测蛋白酶活性。3)与市售ELISA法检测灵敏度的比较。

DESCRIPTION (provided by applicant): Current detection platforms are essentially designed to assay the protein expression level in the biological samples, but for enzymes, not just the expression level but the catalytic activity are also important to their functional role in the pathologic process. We have recently developed a quantum dot-bioluminescence resonance energy transfer (QD-BRET) nanosystem. When a mutant of Renilla luciferase (Luc8) and the QDs are in close proximity, the biochemical energy generated in the oxidation of its substrate, coelenterazine, by Luc8 can be transferred to the QDs through BRET, generating light emission from the QDs. Since this QD-BRET process is distance-dependent, it offers a unique detection platform to assay the enzyme activity. This QD-BRET nanosystem offers many advantages over existing platforms for bioimaging and biodetection. First, by eliminating the need for the external light input, QD-BRET avoids problems associated with fluorescence detection such as background fluorescence, direct acceptor excitation, and photobleaching. Second, it offers great sensitivity for detection. Third, existing detection technologies are subject to the interference from the hemoglobin absorbance, and require serum rather than whole blood analysis. QD-BRET is compatible with analysis of whole blood, thus circumventing the need for serum preparation. Fourth, the QD-BRET platform is amenable to multiplexing and will enable analysis of several targets in a single test. In addition, by avoiding external illumination sources, this technology is much easier to be miniaturized and to be developed into portable diagnostic devices. Fifth, in comparison to the traditional FRET protocols using QDs, QD-BRET uses QD as the emitter which utilizes their outstanding absorption cross-section and broad absorption spectrum. This R21 application seeks to further advance the QD-BRET technology by developing competitive QD- BRET (cQD-BRET) sensors for highly sensitive multiplex detection of enzyme targets in biological samples with three specific aims: 1) Establish the cQD-BRET sensor design for multiplex detection of proteases in a homogenous assay with serum, whole blood and tissue samples. 2) Multiplexed detection of protease activity on a microplate reader platform with immobilized antibodies. 3) Comparison of detection sensitivity with commercial ELISA assay.