Nanotechnology for Multiplex Detection of Enzymes

描述(申请人提供)：目前的检测平台基本上是为了分析生物样本中的蛋白质表达水平而设计的，但对于酶来说，不仅是表达水平，而且催化活性对它们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荧光素酶突变体(Luc8)与量子点非常接近时，Luc8氧化底物coelenterazine产生的生化能量可以通过Bret转移到量子点，从量子点产生光发射。由于这种QD-Bret过程是距离相关的，它提供了一个独特的检测平台来分析酶的活性。与现有的生物成像和生物检测平台相比，这种QD-Bret纳米系统提供了许多优势。首先，通过消除外部光输入的需要，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，它提供了极高的检测灵敏度。第三，现有的检测技术受到血红蛋白吸光度的干扰，需要血清而不是全血分析。QD-Bret与全血分析兼容，从而省去了血清制备的需要。第四，QD-BRET平台可以进行多路传输，可以在一次测试中分析多个目标。此外，由于避免了外部光源，这项技术更容易微型化并开发成便携式诊断设备。第五，与传统的基于量子点的FRET协议相比，QD-Bret采用量子点作为发射体，利用了量子点优异的吸收截面和较宽的吸收光谱。这项R21应用旨在通过开发具有竞争力的QD-Bret(CQD-Bret)传感器来进一步推动QD-Bret技术的发展，该传感器用于高灵敏的多路检测生物样本中的酶靶标，具体目标有三：1)建立CQD-Bret传感器设计，用于在血清、全血和组织样本的均相分析中多重检测蛋白酶。2)在具有固定化抗体的微板阅读器平台上多重检测蛋白水解酶活性。3)检测灵敏度与商品化酶联免疫吸附试验的比较。

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.