Diffusion-Enhanced Lanthanide Nanoparticle FRET Assays

描述(由申请人提供)：我们建议开发一种新的方法来测量生物分子缔合反应。这种方法将基于扩散增强FRET(DE-FRET)。对于纳秒衰减时间，荧光团的扩散基本上不影响FRET的程度。我们将通过使用表面结合的银粒子的稀土氧化物纳米颗粒(LNP)来克服这一限制。每个颗粒和银颗粒中多个稀土离子的存在将弥补典型的低消光和低强度的镧系元素，也避免了增感螯合剂的需要。我们将使用LNPs作为供体和受体标记的检测分子。探测分子与靶的结合将改变受主扩散系数、LNPs强度和衰减时间。DE-FRET数据将揭示平移扩散系数。FCS可以获得类似的信息，但FCS需要仔细控制焦点体积，并且只能在纳摩尔浓度下使用。相比之下，我们的方法不需要特殊的光学元件，适用于任何体积的样品，并且对微摩尔范围内的结合常数敏感，这是与受体结合和药物发现的理想选择。我们将使用两种类型的结合反应来证明我们的方法的有效性。第一个是蛋白质蜂毒素的自结合形成四聚体。蛋白质-蛋白质结合反应的测量对蛋白质组学很重要。第二个将是血清中地高辛的临床检测。这一应用是药物和滥用物质的大类小分子分析的代表。在这个修订的提案中，我们包括了一个重要的生物医学应用，即筛选针对HIV伪病毒或gp120的抗体。这个项目将产生很大的影响，因为该方法可以立即用于一些商业板材阅读器，并很容易添加到大多数现有的板材阅读器。

DESCRIPTION (provided by applicant): We propose to develop a new approach to measuring biomolecule association reactions. This method will be based on diffusion-enhanced FRET (DE-FRET). With nanosecond decay time fluorophores diffusion has essentially no effect on the extent of FRET. We will overcome this limitation by using lanthanide oxide nanoparticles (LNP) which have surface-bound silver particles. The presence of multiple lanthanide ions per particle and Ag particles will compensate for the typical low extinction and intensity of lanthanides, and also circumvent the need for sensitizing chelators. We will use the LNPs as donors and acceptor-labeled detection molecules. Binding of the detection molecules to a target will alter the acceptor diffusion coefficient, the LNPs intensity and decay times. The DE-FRET data will reveal translational diffusion coefficients. Similar information can be obtained by FCS, but FCS requires careful control of the focal volume and can only be used at nanomolar concentrations. In contrast our method will require no special optics, will work with any volume sample and will be sensitive to binding constants in the micromolar range, which is ideal for binding to receptors and drug discovery. We will demonstrate the usefulness of our approach using two types of binding reactions. The first will be self-association of a protein melittin to form tetramer. Measurements of protein- protein association reactions are important for proteomics. The second will be a clinical testing assay of digoxin in serum. This application is representative of large class of small molecule assays of drugs and substances of abuse. In this revised proposal we included an important biomedical application, screening of antibodies against HIV pseudo-virons or gp120. This project will have a high impact because the method can be immediately used with some commercial plate readers and easily added to most existing plate readers.