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

• 批准号: 10596659
• 负责人: Nguyen Ly
• 金额: $ 83.24万
• 依托单位: BIOSENSING INSTRUMENT, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10596659
• 关键词: Acceleration; Address; Arizona; Binding; Binding Proteins; Biological Assay; Biological Models; Biomedical Research; Biosensing Techniques; Cell physiology; Cell surface; Cells; Chemistry; Collaborations; Consumption; Coupled; Detection; Drug Industry; Drug Screening; Drug Targeting; Environment; Exhibits; Feedback; Film; Fluorescence; Glass; Gold; Hela Cells; Image; Imaging Device; Imaging technology; Immobilization; In Situ; Kinetics; Label; Lectin; Ligands; Light; Lighting; Lipids; Mainstreaming; Measurement; Measures; Membrane Glycoproteins; Membrane Proteins; Methods; Molecular; Multiparametric Analysis; Optics; Performance; Pharmaceutical Preparations; Phase; Play; Proteins; Records; Refractive Indices; Resolution; Small Business Technology Transfer Research; Surface; Surface Plasmon Resonance; System; Technology; Testing; Time; United States National Institutes of Health; Universities; Validation; Wheat Germ Agglutinins; Work; absorption; aqueous; biomarker discovery; biomarker validation; commercial prototype; commercialization; cost; detection sensitivity; disease diagnosis; drug candidate; drug development; fluorescence imaging; imaging capabilities; imaging system; improved; instrument; interest; prevent; protein function; prototype; sensor; small molecule; success; temporal measurement; usability

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 总结 测量蛋白质的分子相互作用对于了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，以及用于开发和筛选药物。特别是，委员会认为， 膜蛋白在许多细胞功能中起关键作用，并且是最大类的药物靶标。最 测量膜蛋白相互作用动力学的常用方法包括提取和纯化膜蛋白， 在人工脂质环境中稳定蛋白质，这不仅耗时， 而且劳动密集，而且由于天然细胞微环境的丧失，也可能引入偏差。 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领域， 药物开发CARi建立在表面等离子体共振成像（SPRi）的基础上， 优点，但克服了许多局限性。CARi使用类似于SPRi的光学配置， 测量从感测表面下方反射的光，所述感测表面对诱导的分子结合敏感 折射率在感测表面上方改变。这使得CARi能够检测分子相互作用标记- 免费和实时。然而，与SPRi不同的是，CARi展示了几项独特的技术进步，包括 灵敏度提高~10倍，垂直检测范围提高~100倍，可测量整个细胞表面， 用于正交验证的同时荧光兼容性，更宽的光波长选择， 方便地使用玻璃基表面化学和简单的低成本玻璃传感器芯片。 在这个快速通道STTR项目中，Biosensing Instrument Inc. (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CARi仪器， CARi、SPRi和荧光成像。我们还将在生物医学研究方面与潜在客户合作 和制药行业验证CARi性能和开发关键应用。 该项目的成功将使超高灵敏度的无标记动力学定量的小 单细胞分辨率的膜蛋白上的分子相互作用，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 对于加速生物标志物发现、疾病诊断和药物筛选是非常需要的。