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

用于分子相互作用无标记定量的临界角反射成像

基本信息

批准号：
10641600
负责人：
Nguyen Ly
金额：
$ 28.33万
依托单位：
BIOSENSING INSTRUMENT, INC.
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-15 至 2024-03-31
项目状态：
已结题

项目摘要

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.

标题：临界角反射成像 (CARi) 用于分子相互作用的无标记定量概括测量蛋白质的分子相互作用对于理解蛋白质功能和细胞至关重要过程，用于发现和验证生物标志物以及开发和筛选药物。尤其，膜蛋白在许多细胞功能中发挥关键作用，是最大的一类药物靶点。最多测量膜蛋白相互作用动力学的流行方法包括提取和纯化膜蛋白并在人工脂质环境中稳定蛋白质，这不仅耗时和劳动密集型，但也可能由于天然细胞微环境的丧失而引入偏差。我们建议开发临界角反射成像（CARi）作为原位细胞的突破性技术基于膜蛋白结合相互作用动力学的研究，以推进生物标志物发现和药物开发。 CARi 以表面等离子共振成像 (SPRi) 为基础，获取了其许多独特的功能优点，但克服了许多局限性。 CARi 使用与 SPRi 类似的光学配置测量从传感表面下方反射的光，该表面对诱导的分子结合敏感折射率在传感表面上方发生变化。这使得 CARi 能够检测分子相互作用标记- 免费且实时。然而，与 SPRi 不同的是，CARi 展现了多项独特的技术进步，包括灵敏度提高约 10 倍，测量整个细胞表面的垂直检测范围提高约 100 倍，用于正交验证的同时荧光兼容性、更广泛的光选择波长、方便地使用基于玻璃的表面化学物质，以及简单的低成本玻璃传感器芯片。在这个快速 STTR 项目中，Biosensing Instrument Inc. (BI) 将与 CARi 技术的发明者合作亚利桑那州立大学开发一种商业原型多功能 CARi 仪器，可以执行 CARi、SPRi 和荧光成像。我们还将与生物医学研究领域的潜在客户合作和制药行业验证 CARi 性能并开发关键应用。该项目的成功将为小分子的无标记动力学定量提供超高灵敏度。膜蛋白上的分子相互作用具有单细胞分辨率并允许同时发出荧光用于正交验证的成像。这种基于细胞的无标记原位动力学结合分析的强大功能加速生物标志物发现、疾病诊断和药物筛选非常需要。