An Advanced Biosensor for Molecular Interaction Studies.

描述(申请人提供)：分子相互作用构成生物和化学的基础。它们是生命本身的核心，决定着催化活性，细胞功能， 和治疗效果。绝大多数的诊断程序依赖于某种特定的分子相互作用。因此，在不改变相互作用物种和生理浓度的情况下，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，可用于量化这些互动的工具存在局限性。传统的方法，如蔗糖梯度技术或等温滴定量热法都是费力的，需要大量的样品才能进行分析。荧光和放射性方法是敏感的，但依赖于信号标记的结合来实现检测，这会降低分析速度并增加成本。许多技术，特别是无标记方法，需要将一个相互作用的部分表面固定，将物种置于非本地环境中。标记和系链可能是良性的，但通常会改变相互作用的分子，并可能导致有偏见的结果。最近，我的团队和其他人证明，背向散射干涉法(BSI)可以在学术实验室中用于无标记和自由溶液中的分子相互作用测定，并具有灵敏度，允许在生理相关浓度下对少量样品进行分析。BSI是一种通用的传感方法，它只需要反应产物与参与物种不同地折射光或与光相互作用， 因此，作为分子相互作用光度计(MIP)，具有广泛应用的潜力。BSI是成为MIP的主要候选者，因为它具有简单且廉价的光学序列，包括He-Ne激光器、微流控通道和允许监视微小折射率变化的位置传感器。测量是在玻璃、熔融二氧化硅或塑料中形成的微流控通道内进行的，并在复杂基质(如血清或天然膜蛋白)中以生理相关的浓度进行测量。然而，目前BSI的学术体现在商业上是不可行的。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造成的性能限制，都阻碍了BSI在生命科学和药物研发领域的广泛传播和采用。在这项STTR拨款的第一阶段，我们达到了我们的里程碑，展示了一种双通道BSI仪器，采用了移液管驱动的注射方法和简化了条纹选择和对准的条纹检测算法。在这里，我们建议在这些成果的基础上，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。在第二阶段，我们建议完成四个目标，即通过研究开发和技术转让来完善BSI，促进分子传感公司的商业化，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序列，同时保留了同时执行样品-参考测定的优点。AIM 2实施了一种用户友好的样品引入方法，将污染的可能性降至最低，并将体积限制在1？L。改进的算法提高了灵敏度，实现了电子条纹选择和排列，并解决了非特异性相互作用(在细胞壁)，以提高AIM 3的分析重复性。在Aim 4？-原型版本中，将在Vanderbilt和MSI外部构建和使用BSI系统，以证明BSI提供有意义的定量结合亲和值(从？M到Pm)，并可用于筛选复杂基质中的分子相互作用，如血清和无细胞介质，以及DMSO。在完成这些目标后，将建造三个相同的原型，并对其进行现场应用评估。来自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.