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Interferometric Nano-sensing for Biochemical Analysis

用于生化分析的干涉纳米传感

基本信息

批准号：
7407503
负责人：
DARRYL J. BORNHOP
金额：
$ 19.87万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-01 至 2010-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7407503
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

项目摘要

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-底物的相互作用是理解基本细胞功能和评估治疗的核心。然而，在不修改蛋白质或配体的情况下量化这些相互作用的工具有很大的局限性。底物、受体或蛋白质的表面固定或信号荧光团的附着会影响分子间的相互作用。人们希望消除通常用于结合分析的表面化学，因为它存在着持续存在的问题，包括：由于表面结合靶而导致的复杂的动力学，缺乏耐用性和寿命，高成本，非特异性结合，以及难以对固定的靶进行定量。以MU-TAS格式进行纯液相分子结合分析的能力将消除表面化学产生的许多问题，同时便于对小体积样品进行研究。初步观察表明，分子间相互作用，如蛋白质-蛋白质或配体-底物结合，可以在没有这些扰动的情况下，在高灵敏度下，在皮升体积和自由溶液中使用芯片上干涉后向散射检测(OCIBD)来研究。所提出的技术使用简单且廉价的光学序列来促进这些均匀的、无标签的测量。OCIBD监测在二氧化硅或PDMS中形成的微流体通道内的微小折射率变化。最近，在未经修饰的PDMS通道中进行的OCIBD有助于在没有荧光标记的情况下量化可逆和不可逆的蛋白质-蛋白质结合，直接测量DNA相互作用和对Delta/G的荧光标记扰动。这些测定是在皮升体积和大摩尔水平上进行的，并得到了等温滴定量热仪(ITC)的证实。无标记物的蛋白质-免疫球蛋白在自由溶液中的反应动力学和结合亲和力(Ko)已经可以定量，对免疫球蛋白的检测限为4个阿托莫尔。最近，用背向散射干涉法检测10,800个IL-2分子已经成为可能。这一建议扩展了这些结果，最终形成了用于均相(自由溶液)分子相互作用分析的多路OCIBD。