Collaborative Innovation Center of Chemistry for Energy Materials, the MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Hematology, The First Affiliated Hospital of Xiamen University and Institute of Hematology, School of Medicine, Xiamen University, Xiamen 361005, China.
Department of Laboratory Medicine, Key Laboratory of Clinical Laboratory Technology for Precision Medicine, School of Medical Technology and Engineering Fujian Medical University, Fuzhou 350005, China.
Anal Chem. 2024 Oct 29;96(43):17253-17261. doi: 10.1021/acs.analchem.4c03277. Epub 2024 Oct 20.
Analyzing single-cell protein and mRNA levels yields invaluable insights into cellular functions and the intricacies of biologically heterogeneous systems. Current joint mRNAs and protein analysis methodologies suffer from relative quantification, low sensitivity, possible background interference, and tedious manual manipulation. Therefore, we propose DMF-Bimol that leverages addressable digital microfluidics to automate digital counting of single-cell mRNA and protein based on proximity ligation assay (PLA) and one-step RT-droplet digital PCR (RT-ddPCR). Through an engineered hydrophilic-hydrophobic interface, DMF-Bimol enables efficient single-cell isolation and lossless protein and nucleic acid processing. The closed droplet reaction system enhances the protein concentration and isolates exogenous contaminants, thereby dramatically improving the efficiency of the PLA reaction. The limit of detection of this approach achieves 3313 protein copies, marking a significant 17-fold enhancement in sensitivity over traditional benchtop PLA. This heightened sensitivity also uncovers a lower correlation between mRNA and protein levels in individual cells (Spearman = 0.255) than bulk results, reflecting the complex relationship in heterogeneous cells. Using DMF-Bimol, we observed a significant upsurge of CD147 protein in CD138 myeloma cells but consistent levels of CD147 mRNAs compared with normal leukocytes. This discovery indicates a possible consequence of CD147 oncogenic activation that tends to harness protein translation to bolster tumor cell survival and enhance invasiveness, highlighting the potential of DMF-Bimol in unveiling intricate dynamics in translation processes at the single-cell level.
分析单细胞的蛋白质和 mRNA 水平可以深入了解细胞功能和生物异质系统的复杂性。目前联合的 mRNAs 和蛋白质分析方法存在相对定量、低灵敏度、可能的背景干扰和繁琐的手动操作等问题。因此，我们提出了 DMF-Bimol，它利用可寻址数字微流控技术，根据邻近连接分析（PLA）和一步 RT-液滴数字 PCR（RT-ddPCR）自动对单细胞的 mRNA 和蛋白质进行数字计数。通过工程化的亲水-疏水界面，DMF-Bimol 能够有效地进行单细胞分离，无损地处理蛋白质和核酸。封闭的液滴反应系统可以提高蛋白质浓度并隔离外来污染物，从而显著提高 PLA 反应的效率。该方法的检测限达到 3313 个蛋白质拷贝，比传统的台式 PLA 灵敏度提高了 17 倍。这种更高的灵敏度还揭示了单个细胞中 mRNA 和蛋白质水平之间的相关性较低（Spearman = 0.255），与批量结果相比，这反映了异质细胞中复杂的关系。使用 DMF-Bimol，我们观察到 CD138 骨髓瘤细胞中 CD147 蛋白质的显著增加，但与正常白细胞相比，CD147 mRNAs 的水平保持一致。这一发现表明 CD147 致癌激活可能导致蛋白质翻译增加，以增强肿瘤细胞的存活和侵袭性，突出了 DMF-Bimol 在揭示单细胞水平翻译过程中复杂动态的潜力。
