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.

项目概要 该 R01 应用程序响应 NIH 计划 PAR-19-253“重点技术研究与开发”。 测量受体-配体亲和力的测定在生物医学研究的许多领域都很有价值“金标准”。 表面等离子共振测定仅限于固定在固体表面上的重组可溶性受体。 频率测定（AFA）技术可以测量其天然细胞膜上的受体-配体亲和力。 现有的AFA方法既不能解决单细胞上受体分布不均匀的问题，也不能测量滚动情况 此外，当前的 AFA 方法通常体积大且通量低，这需要 最近，我们发明了一种光驱动微型机器人（LDM）平台，作为一种非侵入式、可编程、 基于这个多功能的LDM平台，我们建议开发一个范例- 移动四维 (4D) AFA（即集成 3D 平移 AFA 和 3D 旋转 AFA）来克服这些关键 在这个 R01 项目中，我们将开发和验证具有以下功能的 4D AFA： (1) 测量天然细胞膜环境上的受体，(2) 解决不均匀分布的受体 在单细胞上，(3) 在集成平台上实现平移和旋转 AFA，(4) 研究细胞粘附 在剪切力和张力下，并且（5）允许进行片上多重细胞粘附测量。 功能，拟议的 4D AFA 有可能超越当前的实验室标准，解决现场未满足的需求，以及 能够以亚细胞分辨率对受体-配体相互作用进行高通量全面分析。我们将验证和改进。 我们将进一步包装和使用经过充分研究的具有可变亲和力的受体-配体对来实现 4D AFA 性能。 应用经过验证的检测方法来研究 SARS-CoV-2 病毒与血管紧张素转换酶 2 受体的结合， 筛选 T 细胞用于巨细胞病毒感染的免疫治疗，在这方面，我们的目标是证明其具有深远的意义。 4D AFA 在改善从临床免疫治疗到基础生物学等领域的研究方面具有潜力。