An Advanced Biosensor for Molecular Interaction Studies.

描述（由申请人提供）：分子相互作用形成生物学和化学的基础。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。绝大多数诊断程序依赖于某种类型的特定分子相互作用。因此，在高灵敏度下进行纯液相分子结合分析的能力，而不改变相互作用的物种，并在生理浓度将是革命性的。然而，可用于量化这些相互作用的工具存在局限性。传统方法（例如蔗糖梯度技术或等温滴定量热法）很费力，并且需要大量样品来进行测定。荧光和放射性方法是敏感的，但依赖于信号标记的掺入来实现检测，这减慢了测定并增加了其成本。许多技术，特别是无标记的方法，需要将物种置于非天然环境中的相互作用部分之一的表面固定化。标签和系链可能是良性的，但通常会改变相互作用的分子，并可能导致有偏见的结果。最近，我的团队和其他人证明，背散射干涉法（BSI）可以在学术实验室中用于进行分子相互作用测定无标记和自由溶液中，灵敏度允许在生理相关浓度下对少量样品进行测定。BSI是一种通用的传感方法，其仅需要反应产物与参与物种不同地对光进行反应或相互作用， 因此，具有作为分子相互作用光度计（MIP）广泛应用的潜力。BSI是成为MIP的主要候选者，因为它具有由He-Ne激光器、微流体通道和位置传感器组成的简单且廉价的光学系统，允许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（如血清或天然膜蛋白）中以生理相关浓度进行。然而，目前BSI的学术实施方案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，我们实现了我们的里程碑，展示了一种双通道BSI仪器，该仪器采用移液器驱动注射方法和条纹检测算法，简化了条纹选择和对齐。在这里，我们建议建立在这些结果的基础上，同时利用两个新的创新，将我们的学术实验室BSI转变为MIP仪器，我们称之为NanoBIND。在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来完善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-参比测定的优点。目标2实现了一种用户友好的样品引入方法，最大限度地减少了污染的可能性，并将体积限制在<1？L.改进的算法提高了灵敏度，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），以提高目标3中的测定重现性。目标4？原型版本BSI系统将被构建和使用，外部的范德比尔特和MSI，以证明BSI给出有意义的和定量的结合亲和力值（从？M至pM），并且其可用于筛选复杂基质如血清和无细胞培养基以及DMSO中的分子相互作用。完成这些目标后，将建造三个相同的原型，并对现场实用性进行评估。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.