Interferometric Nano-sensing for Biochemical Analysis

描述（由申请人提供）：蛋白质-蛋白质和配体-底物相互作用是理解基本细胞功能和评估治疗方法的核心。然而，在不修饰蛋白质或配体的情况下量化这些相互作用的工具有重要的局限性。底物、受体或蛋白质的表面固定化或信号荧光团的附着影响分子间的相互作用。希望消除通常用于结合测定的表面化学的需要，因为它提出了持续存在的问题，包括：由于表面结合目标而导致的复杂动力学，缺乏耐久性和寿命，高成本，非特异性结合以及难以量化固定目标。在mu-TAS格式下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，同时促进小体积样品的研究。本文提出的初步观察表明，分子相互作用，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，以高灵敏度，在皮升体积和自由溶液中使用片上干涉后向散射检测（OCIBD）进行研究。提出的技术使用一种简单而廉价的光学列车来促进这些均匀的，无标签的测量。OCIBD监测在二氧化硅或PDMS形成的微流体通道内的微小折射率变化。在未经修饰的PDMS通道中进行的OCIBD最近促进了可逆和不可逆蛋白质-蛋白质结合的定量，无需荧光标记，直接测量DNA相互作用和荧光标记对δ /G的扰动。这些测定在皮升体积和原子摩尔水平上进行，并通过等温滴定量热法（ITC）确认。在自由溶液中，可以量化无标记蛋白-IgG的反应动力学和结合亲和度（Ko）， IgG的检出限为4个原子。近年来，利用后向散射干涉测量技术已经可以检测到10800个IL-2分子。本提案扩展了这些结果，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。

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.