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

项目摘要 此R 01应用程序响应NIH倡议PAR-19-253“重点技术研究和开发”。 用于测量受体-配体亲和力的测定在生物医学研究的许多领域中是有价值的。“黄金标准” 表面等离子体共振测定限于固定在固体表面上的重组可溶性受体。新出现的粘附 频率测定（AFA）技术可以测量受体-配体在其天然细胞膜上的亲和力。然而，在这方面， 现有的AFA方法既不能解决受体在单个细胞上的非均匀分布， 剪切力下的细胞粘附。此外，当前的AFA方法通常体积庞大且吞吐量低，这需要 繁琐的操作。最近，我们发明了一种光驱动微型机器人（LDM）平台，作为一种非侵入性，可编程， 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例- 移位四维（4D）AFA（即，集成的3D平移AFA和3D旋转AFA）来克服这些关键 现有检测方法的障碍。在这个R 01项目中，我们将开发和验证具有以下功能的4D AFA： (1)测量受体在其天然细胞膜环境中的浓度，（2）分辨非均匀分布的受体 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，和（5）允许芯片上多路复用细胞粘附测量。与这些 功能，拟议的4D AFA有可能超过目前的实验室标准，解决该领域未满足的需求， 能够在亚细胞分辨率下高通量地全面分析受体-配体相互作用。我们将验证和改进 4D AFA的性能，使用充分研究的受体-配体对可变的亲和力。我们将进一步包装， 应用经验证的检测方法研究SARS-CoV-2病毒与血管紧张素转换酶2受体的结合， 筛选巨细胞病毒感染的免疫治疗用T细胞。在这方面，我们的目标是证明 4D AFA的潜力使从临床免疫治疗到基础生物学等领域的研究得以改进。

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.