Four-dimensional Adhesion Frequency Assay for Full Profiling of Receptor-ligand Interactions on Cells

四维粘附频率测定，全面分析细胞上受体-配体相互作用

• 批准号: 10707983
• 负责人: Yuebing Zheng
• 金额: $ 38万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-21 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707983
• 关键词: 2019-nCoV; 3-Dimensional; ACE2; Address; Adhesions; Affinity; Anti-Bacterial Agents; Antiviral Agents; Area; Automation; Bacterial Infections; Benchmarking; Binding; Biological; Biological Assay; Biological Process; Biology; Biomedical Research; Biometry; Biophotonics; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell membrane; Cell surface; Cells; Cellular Membrane; Cellular biology; Clinic; Clinical; Communities; Complex; Cytomegalovirus Infections; Devices; Endothelium; Environment; Epithelial Cells; Event; Four-dimensional; Frequencies; Glycoproteins; Goals; Hematopoietic; Human body; Immunity; Immunotherapy; Infection; Kinetics; Lasers; Ligands; Light; Major Histocompatibility Complex; Maps; Measurement; Measures; Methods; Microfluidics; Modality; Optics; Peptides; Performance; Pharmaceutical Preparations; Process; Production; Recombinants; Research; Research Personnel; Resolution; Rotation; Site; Solid; Stress; Surface; Surface Plasmon Resonance; System; T-Cell Receptor; T-Lymphocyte; Techniques; Technology; Translations; United States National Institutes of Health; Virus; Virus Diseases; austin; biomaterial compatibility; cancer cell; cell fixing; cell killing; clinical application; design; digital; experimental study; imaging system; improved; insight; invention; medical schools; microrobot; microsystems; multimodality; multiplex assay; novel; operation; optical imaging; pathogen; prototype; receptor; recruit; shear stress; simulation; technology research and development; user-friendly

PROJECT SUMMARY This R01 application is responsive to the NIH initiative PAR-19-253 “Focused Technology Research and Development”. Assays for measuring receptor-ligand affinity are valuable in many areas of biomedical research. The “gold standard” surface plasmon resonance assay is limited to recombinant soluble receptors fixed on solid surfaces. The emerging adhesion frequency assay (AFA) techniques can measure the receptor-ligand affinity on their native cellular membranes. However, existing AFA methods can neither resolve the non-uniform distribution of receptors on single cells nor measure the rolling cell adhesion under shear forces. In addition, currentAFAapproaches are generally bulky and low throughput, which require tedious operation. Recently, we have invented a light-driven microrobot (LDM) platform as a non-invasive, programmable, and multimodal cell-manipulation technology. Based on this versatile LDM platform, we propose to develop a paradigm- shift four-dimensional (4D) AFA (i.e., integrated 3D translational AFA and 3D rotational AFA) to overcome these key obstacles in the existing assays. In this R01 project, we will develop and validate our 4D AFA with the following features: (1) measuring receptors on their native cell membrane environments, (2) resolving the non-uniformly distributed receptors on single cells, (3) enabling both translational and rotational AFAs on an integrated platform, (4) investigating cell adhesion under both shear force and tensile force, and (5) allowing on-chip multiplexed cell adhesion measurements. With such features, the proposed 4D AFA has the potential to exceed current lab standards, address unmet needs in the field, and enable high-throughput full profiling of receptor-ligand interactions at sub-cellular resolution. We will validate and improve the 4D AFA performance using well-studied receptor-ligand pairs with variable affinities. We will further package and apply the validated assay to investigate the binding of SARS-CoV-2 virus to angiotensin-converting enzyme 2 receptor and to screen T cells for immunotherapy for cytomegalovirus infection. In this regard, we aim to demonstrate the far-reaching potential of 4D AFA to enable improved research in areas ranging from clinical immunotherapy to fundamental biology.

项目摘要 该R01应用程序对NIH计划响应于19-253 pars“专注的技术研发”。 在生物医学研究的许多领域，测量接收器配体亲和力的测定很有价值。 “黄金标准” 表面等离子体共振测定仅限于固定在固体表面上的重组固体受体。新兴的粘附 频率评估（AFA）技术可以测量其天然细胞膜上的接收器配体亲和力。然而， 现有的AFA方法可以解析单元上接收器的不均匀分布，也可以测量滚动 剪切力下的细胞粘合剂。此外，Currentafaappracties通常是笨重且低吞吐量的，这需要 乏味的操作。最近，我们发明了一个轻驱动的微型机器人（LDM）平台，是一种无创，可编程的，可编程的， 和多模式细胞操作技术。基于这个多功能LDM平台，我们建议开发一个范式 - 移动四维（4D）AFA（即集成的3D转换AFA和3D旋转AFA）以克服这些键 现有阿萨斯的障碍。在此R01项目中，我们将使用以下功能开发和验证我们的4D AFA： （1）测量其本地细胞膜环境上的接收器，（2）解决不均匀分布的接收器 在单个单元格上，（3）在集成平台上启用翻译和旋转AFA，（4）研究细胞粘附 在剪切力和拉伸力下，（5）允许片上多重细胞粘合剂测量。这样 功能，拟议的4D AFA有可能超过当前的实验室标准，解决现场未满足的需求以及 在亚细胞分辨率下实现受体配体相互作用的高通量完整分析。我们将验证和改进 使用良好的接收器配体对具有可变亲和力的4D AFA性能。我们将进一步打包 应用经过验证的评估来研究SARS-COV-2病毒与血管紧张素转换酶2受体的结合和 筛选T细胞进行免疫疗法以进行巨细胞病毒感染。在这方面，我们旨在展示深远的角度 4D AFA的潜力可以改善从临床免疫疗法到基本生物学的领域的研究。