Diffusion-Enhanced Lanthanide Nanoparticle FRET Assays

扩散增强型镧系元素纳米粒子 FRET 测定

• 批准号: 9095386
• 负责人: Joseph R. LAKOWICZ
• 金额: $ 19.19万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9095386
• 关键词: Affect; Amino Acids; Antibodies; Antigens; Bee Venoms; Binding; Biological; Biological Assay; Blood; Capsid Proteins; Chelating Agents; Clinical; Collaborations; Complex; Computer software; Data; Detection; Diagnostic; Diffuse; Diffusion; Digoxin; Drug Receptors; Electronics; Epitopes; Extinction (Psychology); Fab Immunoglobulins; Fluorescence; Fluorescence Polarization Immunoassay; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Generic Drugs; Goals; Gold; HIV; HIV Antibodies; HIV Envelope Protein gp120; Health; Human; Institutes; Ions; Label; Lanthanoid Series Elements; Lasers; Light; Measurement; Measures; Medical; Membrane; Metals; Methods; Microscope; Modeling; Molecular; N-terminal; Optics; Oxides; Particle Size; Peptides; Pharmaceutical Preparations; Proteins; Proteomics; Reaction; Reader; Rhodamine; Sampling; Scanning; Sensitivity and Specificity; Serum; Silver; Source; Spectrum Analysis; Staging; Substance of Abuse; Surface; System; Testing; Therapeutic; Time; Vaccines; Virion; Work; absorption; base; biological research; drug discovery; fluorophore; macromolecule; millisecond; nanomolar; nanoparticle; nanosecond; neutralizing antibody; novel; novel strategies; particle; receptor; research clinical testing; screening; small molecule; virology; viron

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.

描述（由申请人提供）：我们建议开发一种测量生物分子缔合反应的新方法。该方法将基于扩散增强 FRET (DE-FRET)。在纳秒衰减时间下，荧光团扩散对 FRET 的程度基本上没有影响。我们将通过使用具有表面结合银颗粒的镧系氧化物纳米颗粒（LNP）来克服这一限制。每个粒子和银粒子中存在多个镧系元素离子将补偿镧系元素典型的低消光和强度，并且还避免了对敏化螯合剂的需要。我们将使用 LNP 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、LNP 强度和衰减时间。 DE-FRET 数据将揭示平移扩散系数。通过FCS可以获得类似的信息，但是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，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，这对于与受体结合和药物发现来说是理想的。我们将使用两种类型的结合反应来证明我们的方法的有用性。第一个是蜂毒素蛋白自缔合形成四聚体。蛋白质-蛋白质关联反应的测量对于蛋白质组学很重要。第二个是血清中地高辛的临床检测。该应用代表了一大类滥用药物和物质的小分子测定。在这个修订后的提案中，我们纳入了一项重要的生物医学应用，即针对 HIV 假病毒或 gp120 的抗体的筛选。该项目将产生很大的影响，因为该方法可以立即与一些商业读板器一起使用，并且可以轻松添加到大多数现有的读板器中。