An Advanced Biosensor for Molecular Interaction Studies

描述（由申请人提供）：分子相互作用是理解基础生物学和细胞功能的核心，对所有分子分析（研究和体外诊断）至关重要，在生物标志物发现中至关重要，对评估治疗方法至关重要。然而，量化这些相互作用的工具有局限性，因为它们要么依赖于通过结合标记方案来改变相互作用分子的天然状态，要么要求其中一个相互作用部分的表面固定化，要么需要大量的样品。因此，在小型化的情况下，以高灵敏度进行纯液相分子结合分析的能力是理想的，现在已经用后向散射干涉测量（BSI）证明了这一点。BSI采用一种简单而廉价的光学系统，由氦氖激光器、微流体通道和监测微小折射率变化的位置传感器组成。这些测量可以在玻璃、熔融二氧化硅或塑料中形成的微流控通道内进行，并在复杂基质中以生理相关浓度进行。然而，目前BSI的体现是有限的。繁琐的比对方法、不成熟的转导方案、不完善的样品处理和导入方法、昂贵的一次性芯片以及环境噪声敏感性导致的性能限制，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。我们建议改进BSI，促进分子传感公司的商业化，并允许随后在生物研究界广泛传播（在未来的第二阶段拨款下）。在这个第一阶段的项目中，我们将通过实现三个具体目标来实现向工业部门的转移：1)构建一个光学简单的双通道BSI，以提高信噪比和环境噪声补偿；2)通过使用基于相互关联的位置感知算法简化对准和提高性能；3)实现用户友好的图形界面（GUI）。模型相互作用系统将用于证明BSI给出有意义和定量的结合亲和力值（从<M到pM），并且它可用于筛选各种基质（血清、无细胞培养基、DMSO）中的分子相互作用。

DESCRIPTION (provided by applicant): Molecular interactions are central to the understanding of basic biology and cellular function, are paramount to all molecular assays (research and in vitro diagnostics), are essential in biomarker discovery and critical toward evaluating therapeutics. However, the tools available to quantify these interactions have limitations, in so far as they either rely upon altering the native state of the interacting molecules by incorporating labeling schemes, demanding surface immobilization of one of the interacting moieties or require prohibitive quantities of sample. The ability to perform pure liquid-phase molecular binding analysis at high sensitivity, in a miniaturized format, is therefore desirable and has now been demonstrated with back-scattering interferometry (BSI). BSI employs a simple and inexpensive optical train comprised of a He-Ne laser, a microfluidic channel, and a position sensor monitoring minute refractive index changes. These measurements may be made within a microfluidic channel formed in glass, fused silica, or plastic and at physiologically relevant concentrations in complex matrices. Yet the current embodiment of BSI is limited. Tedious alignment methods, immature transduction schemes, poorly refined sample handling and introduction methods, expensive disposable chips, 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. We propose to refine BSI, facilitating commercialization by Molecular Sensing Inc. and allowing the subsequent broad dissemination in the biological research community (under a future Phase II grant). Under this Phase I project, we will enable transfer to the industrial sector by performing three specific aims: 1) constructing an optically simple two channel BSI for enhanced S/N and environmental noise compensation, 2) simplifying alignment and improving performance through the use of a cross-correlation based position sensing algorithm and 3) implementing a user friendly graphical interface (GUI). Model interaction systems will be used 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 various matrices (serum, cell-free media, DMSO). PUBLIC HEALTH RELEVANCE: Proteins interact constantly with one another and with other entities in the cellular and extracellular environment. However, sensitive in vitro methods to monitor such interactions, in particular methods that are label-free and do not involve surface immobilization, have until recently been lacking. Free-solution, label-free, molecular interactions can now be investigated using back-scattering interferometry (BSI), allowing quantification of KD values ranging from micromolar to picomolar using miniscule quantities of the binding pairs. With BSI, little a priori knowledge about the proteins, antibodies, or drug targets etc. is necessary to quantify the level of interaction and screen for efficacy or perform a diagnosis. The BSI methodology has the potential to shift the paradigm for molecular interaction studies, allowing screening for unknown binding partners of orphan receptors, study of inhibitors for GPCR targets, as well as for numerous other binding pairs and is likely applicable to diagnostics and therapeutic monitoring in human specimens. Furthermore, the technology is simple and inexpensive allowing for the potential of BSI to be widely disseminated.