Critical angle reflection imaging for label-free quantification of molecular interactions

标题： 临界角反射成像用于分子相互作用的无标记定量 摘要 测量蛋白质的分子相互作用对于了解蛋白质的功能和细胞 生物标记物的发现和验证，以及药物的开发和筛选。特别是， 膜蛋白在许多细胞功能中起着关键作用，是最大的药物靶点。多数 常用的测量膜蛋白相互作用动力学的方法包括提取和纯化 膜蛋白和在人造脂质环境中稳定蛋白质，这不仅耗时 和劳动密集型，但也可能会引入偏见，因为失去了天然的细胞微环境。 我们提出了发展临界角反射成像(CARI)作为原位细胞成像的突破性技术。 基于膜蛋白结合动力学的研究，以促进生物标志物的发现和领域的发展 药物开发。CAI建立在表面等离子体共振成像(SPRI)的基础上，获得了许多独特的 优势，但克服了它的许多局限性。CARI使用类似于SPRI的光学配置， 测量从传感表面下方反射的光，该光对分子结合很敏感 传感面上方的折射率发生变化。这使得CAI能够检测分子相互作用标记- 免费和实时的。然而，与SPRI不同的是，CAI展示了几个明显的技术进步，包括 ~10倍的灵敏度，~100倍的垂直探测范围，用于测量整个细胞表面， 用于正交验证的同时荧光兼容性、更宽的光波长选择、 使用方便的玻璃基表面化学物质，以及简单的低成本玻璃传感器芯片。 在这个快速通道STTR项目中，生物传感仪器公司(BI)将与CARI技术的发明者合作 在亚利桑那州立大学开发一种商业原型多功能CAI仪器，可以执行 CARI、SPRI和荧光成像。我们还将与潜在客户在生物医学研究方面进行合作 和制药行业，以验证CAI性能并开发关键应用。 该项目的成功将使无标记动力学定量的超高灵敏度成为可能 膜蛋白上的分子相互作用具有单细胞分辨率并允许同时荧光 用于正交验证的成像。这种强大的无标记原位细胞动力学结合分析能力 对于加快生物标记物的发现、疾病诊断和药物筛选是非常必要的。

TITLE: Critical angle reflection imaging (CARi) for label-free quantification of molecular interactions SUMMARY Measuring molecular interactions of proteins are critical for understanding protein functions and cellular processes, for discovery and validating biomarkers, and for developing and screening drugs. In particular, membrane proteins play key roles in many cellular functions and are the largest class of drug targets. Most popular methods for measuring membrane protein interaction kinetics involves extraction and purification of membrane proteins and stabilizing the proteins in an artificial lipid environment, which is not only time consuming and labor intensive, but also may introduce bias due to the loss of the native cellular microenvironment. We proposed to develop critical angle reflection imaging (CARi) as a breakthrough technology for in-situ cell- based studies of membrane protein binding interaction kinetics to advance the field of biomarker discovery and drug development. CARi builds upon surface plasmon resonance imaging (SPRi), acquiring many of its unique advantages, but overcoming many of its limitations. CARi uses an optical configuration similar to SPRi that measures light reflected from below the sensing surface, which is sensitive to molecular bindings induced refractive index changes above the sensing surface. This enables CARi to detect molecular interaction label- free and in real-time. However, unlike SPRi, CARi exhibits several distinct technological advances, including a ~10 times greater sensitivity, ~100 times greater vertical detection range for measuring entire cell surfaces, simultaneous fluorescence compatibility for orthogonal validation, broader wavelength of light selection, convenient use of glass-based surface chemistries, and simple low-cost glass sensor chips. In this fast-track STTR project, Biosensing Instrument Inc. (BI) will work with the inventor of CARi technology at Arizona State University to develop a commercial prototype multi-functional CARi instrument that can perform CARi, SPRi, and fluorescence imaging. We will also collaborate with potential customers in biomedical research and pharmaceutical industries to validate CARi performance and develop key applications. The success of this project will enable ultra-high sensitivity for label-free kinetic quantification of small molecule interactions on membrane proteins with single-cell resolution and permit simultaneous fluorescence imaging for orthogonal validation. This powerful capability of label-free in-situ cell-based kinetic binding analysis is greatly needed for expediting biomarker discovery, disease diagnosis and drug screening.