Diffusion-Enhanced Lanthanide Nanoparticle FRET Assays

描述（由申请人提供）：我们建议开发一种测量生物分子结合反应的新方法。这种方法将基于扩散增强FRET （DE-FRET）。随着纳秒级的衰减时间，荧光团的扩散基本上对FRET的程度没有影响。我们将通过使用表面结合银粒子的氧化镧纳米粒子（LNP）来克服这一限制。每个粒子和银粒子的多个镧系离子的存在将补偿镧系元素典型的低消光和强度，也规避了敏化螯合剂的需要。我们将使用LNPs作为供体和受体标记的检测分子。检测分子与目标的结合将改变受体扩散系数、LNPs强度和衰减时间。DE-FRET数据将揭示平动扩散系数。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器件，可以处理任何体积的样品，并且对微摩尔范围内的结合常数敏感，这对于结合受体和药物发现是理想的。我们将用两种类型的结合反应来证明我们的方法的有效性。首先是一种蛋白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非常重要。第二项将是血清中地高辛的临床检测。该应用是一类具有代表性的小分子分析药物和滥用物质。在这个修改后的提案中，我们包括了一个重要的生物医学应用，HIV伪病毒或gp120抗体的筛选。这个项目将有很大的影响，因为该方法可以立即与一些商业车牌阅读器一起使用，并且很容易添加到大多数现有的车牌阅读器中。

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.