Nanotechnology for Multiplex Detection of Enzymes

描述（由申请人提供）：目前的检测平台基本上被设计用于测定生物样品中的蛋白质表达水平，但对于酶，不仅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。当海肾荧光素酶（Luc 8）的突变体和QD紧密接近时，Luc 8氧化其底物腔肠素时产生的生化能量可以通过BRET转移到QD，从而从QD产生光发射。由于这种QD-BRET过程是距离依赖性的，它提供了一个独特的检测平台来测定酶的活性。这种QD-BRET纳米系统提供了许多优于现有生物成像和生物检测平台的优势。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，例如背景荧光、直接受体激发和光漂白。其次，它提供了很高的检测灵敏度。第三，现有的检测技术受到来自血红蛋白吸光度的干扰，并且需要血清而不是全血分析。QD-BRET与全血分析兼容，因此无需血清制备。第四，QD-BRET平台适合于多路复用，并且将能够在单个测试中分析几个目标。此外，由于避免了外部照明源，该技术更容易被小型化并被开发成便携式诊断设备。第五，与使用QD的传统FRET方案相比，QD-BRET使用QD作为发射体，其利用其突出的吸收截面和宽吸收光谱。该R21申请寻求通过开发用于生物样品中的酶靶标的高灵敏度多重检测的竞争性QD-BRET（cQD-BRET）传感器来进一步推进QD-BRET技术，具有三个特定目标：1）建立用于在具有血清、全血和组织样品的均质测定中多重检测蛋白酶的cQD-BRET传感器设计。2)用固定化抗体在酶标仪平台上多重检测蛋白酶活性。3)与商业ELISA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.