Interferometric Nano-sensing for Biochemical Analysis

用于生化分析的干涉纳米传感

• 批准号: 7226192
• 负责人: DARRYL J. BORNHOP
• 金额: $ 19.84万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7226192
• 关键词: Affinity; Binding; Biochemical; Biological; Biological Assay; Calorimetry; Cell physiology; Chemistry; Compatible; Consumption; DNA; Detection; Development; Enzymes; Equilibrium; Financial compensation; Generations; Immobilization; Immunoglobulin G; Interferometry; Interleukin-2; Investigation; Kinetics; Label; Ligand Binding; Ligands; Liquid substance; Longevity; Measurement; Methodology; Microfluidics; Modeling; Molecular; Monitor; Noise; Optics; Performance; Phase; Procedures; Process; Protein Binding; Proteins; Proteolysis; Reaction; Reagent; Refractive Indices; Research; Research Personnel; Sampling; Signal Transduction; Silicon Dioxide; Siloxanes; Solutions; Source; Substrate Interaction; Surface; Surface Plasmon Resonance; System; Techniques; Technology; Theoretical model; Therapeutic; Thermodynamics; Titrations; Training; analog; base; cost; detector; fluorophore; high throughput screening; improved; instrument; interest; macromolecule; micro-total analysis system; nanoscale; nanosensors; programs; protein protein interaction; receptor; sensor; solute; tool

DESCRIPTION (provided by applicant): Protein - protein and ligand - substrate interactions are central to understanding basic cellular function and for evaluating therapeutics. However, the tools available to quantify these interactions without modifying the proteins or ligands have important limitations. Surface immobilization of the substrate, receptor, or protein or the attachment of a signaling fluorophore influences molecular interactions. It is desirable to eliminate the need for the surface chemistry normally used in binding assays, since it presents persistent problems including: complicated kinetics due to surface bound targets, lack of durability and longevity, high cost, non-specific binding, and difficulty in quantification of the immobilized targets. The ability to perform pure liquid - phase molecular binding analysis in a mu-TAS format would eliminate many of the problems that arise from the surface chemistry, while facilitating small volume samples to be studied. Preliminary observations presented here indicate that molecular interactions, such as protein - protein or ligand - substrate binding, can be studied in the absence of these perturbations, at high sensitivity, in picoliter volumes and in free-solution using on-chip interferometric backscatter detection (OCIBD). The proposed technology uses a simple and inexpensive optical train to facilitate these homogeneous, label-free measurements. OCIBD monitors minute refractive index changes within a microfluidics channel formed in silica or PDMS. OCIBD performed in a non-modified PDMS channel has recently facilitated quantification of reversible and irreversible protein - protein binding without a fluorescent label, the direct measurement of DNA interactions and fluorescent tag perturbations on delta/G. These determinations were performed in picoliter volumes and at attomolar levels and confirmed by Isothermal Titration Calorimetry (ITC). It has been possible to quantify reaction kinetics and binding affinities (Ko) for protein-IgG label-free in free-solution, with detection limits of 4 attomoles for IgG. Recently it has been possible to detect 10,800 molecules of IL-2 with backscattering interferometry. This proposal expands on these results, culminating a multiplexed OCIBD for homogeneous (free-solution) molecular interaction assays.

描述（由申请人提供）：蛋白质 - 蛋白质和配体 - 底物相互作用对于理解基本细胞功能和评估治疗剂至关重要。但是，可用于量化这些相互作用的工具而不修改蛋白质或配体具有重要的局限性。底物，受体或蛋白质或信号传导荧光团的附着的表面固定会影响分子相互作用。希望消除通常用于结合测定法的表面化学的需求，因为它提出了持续的问题，包括：由于表面结合目标，缺乏耐用性和寿命，高成本，非特异性结合以及量化固定目标的量化难度。以MU -TAS格式进行纯液体 - 相分子结合分析的能力将消除由表面化学产生的许多问题，同时促进要研究的小体积样品。此处介绍的初步观察表明，在没有这些扰动的情况下，在高灵敏度，PICOLITER体积和自由溶液中，可以使用芯片干涉量表后粒子检测（OCIBD）来研究分子相互作用，例如蛋白质 - 蛋白质或配体 - 底物结合。提出的技术使用简单且廉价的光学列车来促进这些均匀的无标签测量。 OCIBD监视二氧化硅或PDM形成的微流体通道内的折射率变化。在非修饰的PDMS通道中进行的OCIBD最近促进了对无荧光标记的可逆蛋白质 - 蛋白质结合的定量，直接测量DNA相互作用和荧光标签在delta/g上的荧光扰动。这些确定是在Picoliter的体积和attomolar水平上进行的，并通过等温滴定量热法（ITC）确认。可以量化反应动力学和结合亲和力（KO），以在自由溶液中无蛋白质标记，而IgG的检测限为4个attomoles。最近，可以通过反向散射干涉法检测10,800个IL-2分子。该建议扩展了这些结果，最终导致多重OCIBD用于均质（自由分析）分子相互作用测定。