An Advanced Biosensor for Molecular Interaction Studies.

描述（由申请人提供）：分子相互作用构成了生物学和化学的基础。它们是生命本身的核心，决定催化活性、细胞功能、 和治疗效果。绝大多数诊断程序依赖于某种类型的特定分子相互作用。因此，在不改变相互作用的物质的情况下，在生理浓度下以高灵敏度进行纯液相分子结合分析的能力将是革命性的。然而，可用于量化这些相互作用的工具存在局限性。蔗糖梯度技术或等温滴定量热法等传统方法非常费力，并且需要大量样品才能进行测定。荧光和放射性方法很敏感，但依赖于信号标记的结合来实现检测，从而减慢了测定速度并增加了成本。许多技术，特别是无标记方法，需要对相互作用部分之一进行表面固定，从而将物种置于非天然环境中。标签和系链可能是良性的，但通常会改变相互作用的分子，并可能导致有偏差的结果。最近，我的团队和其他人证明，背向散射干涉测量 (BSI) 可在学术实验室中用于无标记和自由溶液中进行分子相互作用测定，其灵敏度允许在生理相关浓度下对少量样品进行测定。 BSI 是一种通用传感方法，只需要反应产物以不同于参与物种的方式折射或与光相互作用， 因此有可能作为分子相互作用光度计（MIP）广泛应用于一般用途。 BSI 是成为 MIP 的主要候选者，因为它具有简单且廉价的光学系统，由氦氖激光器、微流体通道和位置传感器组成，可以监测微小的折射率变化。测量是在玻璃、熔融石英或塑料中形成的微流体通道内进行的，并且在复杂基质（例如血清）或天然膜蛋白中以生理相关浓度进行。然而，目前 BSI 的学术体现在商业上并不可行。繁琐的比对方法、不成熟的转导方案、不完善的样品处理和引入方法以及由于环境噪声敏感性而导致的性能限制都阻碍了 BSI 在生命科学和药物发现领域的广泛传播和采用。在 STTR 资助的第一阶段，我们实现了里程碑，展示了双通道 BSI 仪器，采用移液器驱动注射方法和简化条纹选择和对齐的条纹检测算法。在这里，我们建议以这些成果为基础，同时利用两项新的创新，将我们的学术实验室 BSI 转变为我们称为 NanoBIND 的 MIP 仪器。根据 STTR 第二阶段，我们建议完成四个目标，通过研究开发和技术转让完善 BSI，促进 Molecular Sensing Inc. 的商业化，并允许随后在研究界广泛传播。在目标 1 中，我们进一步简化了光学系统，同时保留了同时进行样品参考测定的优点。目标 2 实施了一种用户友好的进样方法，最大限度地减少了污染的可能性并将体积限制在 <1?L。改进的算法增强了灵敏度，实现了电子条纹选择和对齐，并解决了非特异性相互作用（在细胞壁处），以提高 Aim 3 中的测定重现性。在 Aim 4 中，将在 Vanderbilt 和 MSI 外部构建和使用原型版 BSI 系统，以证明 BSI 提供有意义的定量结合亲和力值（从 ?M 到 pM），并且它也可用于筛选复杂基质（例如血清和无细胞培养基）中的分子相互作用。如二甲基亚砜。完成这些目标后，将建造三个相同的原型并评估其现场实用性。这些实验室和用户的反馈将提供正式的 在第三阶段商业部署下完善设计的框架。

DESCRIPTION (provided by applicant): Molecular interactions form the foundation of biology and chemistry. They are central to life itself and determine catalytic activity, cellular function, and therapeutic efficacy. The vast majority of diagnostic procedures depend on some type of specific molecular interaction. Therefore, the ability to perform pure liquid-phase molecular binding analysis at high sensitivity, without modifying the interacting species, and at physiological concentrations would be revolutionary. Yet, the tools available to quantify these interactions have limitations. Traditional methods such as the sucrose gradient technique or isothermal titrimetric calorimetry are laborious and require substantial quantities of sample to perform an assay. Fluorescence and radioactive methods are sensitive, but rely on the incorporation of signaling labels to enable detection, slowing the assay and increasing its cost. Numerous techniques, particularly the label-free methods, require surface immobilization of one of the interacting moieties putting the species in a non-native environment. Labels and tethers can be benign, but often alter the interacting molecules and can lead to a biased result. Recently my group and others demonstrated that back-scattering interferometry (BSI) can be used in the academic laboratory to perform molecular interaction determinations label-free and in free-solution, with sensitivity that allows assays on small quantities of sample, at physiologically relevant concentrations. BSI is a universal sensing method that only requires the product of a reaction to refract or interact with light differently than the participating species, therefore has the potential to be widely applicable for general use as a Molecular Interaction Photometer (MIP). BSI is a prime candidate to become an MIP because it has a simple and inexpensive optical train comprised of a He-Ne laser, a microfluidic channel, and a position sensor allowing minute refractive index changes to be monitored. Measurements are made within a microfluidic channel formed in glass, fused silica, or plastic and at physiologically relevant concentrations in complex matrices such as serum or with native membrane- proteins. Yet the current academic embodiment of BSI is not commercially viable. Tedious alignment methods, immature transduction schemes, poorly refined sample handling and introduction methods, and performance limitations due to environmental noise sensitivity all impede the wide dissemination and adoption of BSI in the life science and drug discovery communities. Under Phase I of this STTR grant we met our milestones demonstrating a two-channel BSI instrument with a pipette-driven injection method and a fringe detection algorithm that simplified fringe selection and alignment. Here we propose to build on these results, while capitalizing on two new innovations to transform our academic laboratory BSI into the MIP instrument we call NanoBIND. Under this STTR Phase II, we propose the completion of four aims to refine BSI through research development and technology transfer, facilitate commercialization by Molecular Sensing Inc. and allow the subsequent broad dissemination in the research community. In Aim 1 we further simplify the optical train, while retaining the advantages of performing a simultaneous sample-reference determination. Aim 2 implements a sample introduction method that is user friendly, minimizes the potential for contamination and constrains volumes to <1?L. An improved algorithm enhances sensitivity, enables electronic fringe selection and alignment and addresses non-specific interactions (at cell wall) to improve assay reproducibility in Aim 3. And in Aim 4 ?- prototype version BSI systems will be constructed and used, external to Vanderbilt and MSI, to demonstrate that BSI gives meaningful and quantitative binding affinity values (from ?M to pM) and that it can be used to screen for molecular interactions in complex matrices such as serum and cell-free media, as well as DMSO. Upon completion of these Aims, three identical prototypes will have been constructed and evaluated for field utility. Feedback from these laboratories and users will provide the formal framework for refining the design under Phase III commercial deployment.