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

项目总结 此R01应用程序响应美国国立卫生研究院PAR-19-253“重点技术研究和开发”倡议。 测定受体-配体亲和力的分析方法在生物医学研究的许多领域都很有价值。“黄金标准” 表面等离子体共振检测仅限于固定在固体表面的重组可溶性受体。新出现的粘附力 频率分析(AFA)技术可以测量其天然细胞膜上的受体-配体亲和力。然而， 现有的AFA方法既不能解决受体在单个细胞上的不均匀分布，也不能测量细胞上的滚动 剪切力作用下的细胞黏附。此外，目前的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、可编程的 以及多模式细胞操纵技术。基于这个通用的LDM平台，我们建议开发一个范例-- 移动四维(4D)AFA(即集成的3D平移AFA和3D旋转AFA)以克服这些关键 现有检测方法中存在的障碍。在这个R01项目中，我们将开发并验证我们的4D AFA，具有以下功能： (1)测量天然细胞膜环境中的受体；(2)解析分布不均匀的受体 在单个细胞上，(3)在集成平台上启用平移和旋转AFA，(4)研究细胞黏附 可同时承受剪切力和拉力，以及(5)允许进行片上多路细胞黏附测量。有了这样的 功能，拟议的4D AFA有可能超过当前的实验室标准，解决该领域未得到满足的需求，以及 以亚细胞分辨率实现高通量的受体-配体相互作用的全谱分析。我们将进行验证和改进 使用研究良好的具有可变亲和力的受体-配体对的4D AFA性能。我们将进一步打包和 应用验证的方法研究SARS-CoV-2病毒与血管紧张素转换酶2受体和 为巨细胞病毒感染的免疫治疗筛选T细胞。在这方面，我们旨在展示影响深远的 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善。

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.