Department of Biomedical Engineering, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong SAR 999077, China.
City University of Hong Kong Shenzhen Research Institute, Shenzhen Virtual University Park, Shenzhen 518057, China.
Anal Chem. 2024 Jan 9;96(1):49-58. doi: 10.1021/acs.analchem.3c02562. Epub 2023 Dec 18.
Single-cell multiplexed phenotypic analysis expands the biomarkers for diagnosis, heralding a new era of precision medicine. Cell secretions are the primary measures of immune function, but single-cell screening remains challenging. Here, a novel cell membrane-based assay was developed using cholesterol-linked antibodies (CLAbs), integrating immunosorbent assays and droplet microfluidics to develop a flexible high-throughput single-cell secretion assay for multiplexed phenotyping. CLAb-grafted single cells were encapsulated in water-in-oil droplets to capture their own secretions. Subsequently, the cells were extracted from droplets for fluorescence labeling and screening. Multiple secretions and surface proteins were simultaneously measured from single cells by flow cytometry. To validate the approach, THP-1 cells, THP-1-derived M1 macrophages, and dendritic cells were assayed, indicating the differentiation efficiency of THP-1 cells under different chemical stimulations. Moreover, peripheral blood mononuclear cells from healthy donors under various stimuli showed varied active immune cell populations (6.62-47.14%). The peripheral blood mononuclear cells (PBMCs) of nasopharyngeal carcinoma patients were analyzed to identify a higher percentage of actively cytokine-secreted single cells in the basal state (2.82 ± 1.48%), compared with that in the health donors (0.70 ± 0.29%).
单细胞多重表型分析扩展了用于诊断的生物标志物，开创了精准医学的新纪元。细胞分泌物是免疫功能的主要衡量标准，但单细胞筛选仍然具有挑战性。在这里，开发了一种使用胆固醇连接抗体（CLAbs）的新型细胞膜分析方法，该方法集成了免疫吸附测定和液滴微流控技术，开发了一种灵活的高通量单细胞分泌分析方法，用于多重表型分析。将接枝了 CLAb 的单个细胞包裹在油包水液滴中以捕获其自身的分泌物。随后，从液滴中提取细胞进行荧光标记和筛选。通过流式细胞术可同时从单个细胞中测量多种分泌物和表面蛋白。为了验证该方法，对 THP-1 细胞、THP-1 衍生的 M1 巨噬细胞和树突状细胞进行了测定，表明了不同化学刺激下 THP-1 细胞的分化效率。此外，来自健康供体的外周血单核细胞在各种刺激下显示出不同的活性免疫细胞群（6.62-47.14%）。分析了鼻咽癌患者的外周血单核细胞，以鉴定在基础状态下具有更高比例的主动细胞因子分泌单细胞（2.82±1.48%），而健康供体中为（0.70±0.29%）。
