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: 10325802
• 负责人: Nguyen Ly
• 金额: $ 25.47万
• 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 2022-02-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10325802
• 关键词: Address; Arizona; Binding; Binding Proteins; Biological Assay; Biological Markers; Biological Models; Biomedical Research; Biosensing Techniques; Cell physiology; Cell surface; Cells; Chemistry; 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; base; biomarker discovery; 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），用于无标记的分子相互作用 概括 测量蛋白质的分子相互作用对于理解蛋白质和细胞至关重要 过程，发现和验证生物标志物以及用于开发和筛查药物。尤其， 膜蛋白在许多细胞功能中起关键作用，并且是最大的药物靶标。最多 测量膜蛋白相互作用动力学的流行方法涉及提取和纯化 膜蛋白质并在人造脂质环境中稳定蛋白质，这不仅是耗时的 和劳动密集型，但由于本地细胞微环境的丧失，也可能引起偏见。 我们提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，但克服了许多局限性。 Cari使用类似于Spri的光学配置 测量从感应表面下方反射的光，该光对分子结合敏感 折射率在传感表面上方变化。这使Cari能够检测分子相互作用标记 - 免费，实时。但是，与Spri不同，Cari展示了几种不同的技术进步，包括 左右灵敏度的〜10倍，大约要测量整个细胞表面的垂直检测范围约100倍 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和简单的低成本玻璃传感器芯片。 在这个快速轨道的STTR项目中，BioSensing Instrument Inc.（BI）将与Cari Technology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，可以执行 Cari，Spri和荧光成像。我们还将与潜在客户合作生物医学研究 和制药行业，以验证CARI绩效并开发关键应用。 该项目的成功将使小型无标签的动力学定量实现超高灵敏度 具有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 对正交验证的成像。这种无标签基于原位细胞的动力学动力学结合分析的强大能力 加快生物标志物发现，疾病诊断和药物筛查非常需要。