PDF(Pubmed)
Abstract：
Archived patient-derived tissue specimens play a central role in understanding disease and developing therapies. To address specificity and sensitivity shortcomings of existing single-cell resolution proteoform analysis tools, we introduce a hybrid microfluidic platform (DropBlot) designed for proteoform analyses in chemically fixed single cells. DropBlot serially integrates droplet-based encapsulation and lysis of single fixed cells, with on-chip microwell-based antigen retrieval, with single-cell western blotting of target antigens. A water-in-oil droplet formulation withstands the harsh chemical (SDS, 6 M urea) and thermal conditions (98 °C, 1-2 hr) required for effective antigen retrieval, and supports analysis of retrieved protein targets by single-cell electrophoresis. We demonstrate protein-target retrieval from unfixed, paraformaldehyde-fixed (PFA), and methanol-fixed cells. Key protein targets (HER2, GAPDH, EpCAM, Vimentin) retrieved from PFA-fixed cells were resolved and immunoreactive. Relevant to biorepositories, DropBlot profiled targets retrieved from human-derived breast tumor specimens archived for six years, offering a workflow for single-cell protein-biomarker analysis of sparing biospecimens.
摘要：
存档的患者来源的组织标本在了解疾病和开发治疗中起着核心作用。为了解决现有单细胞解析蛋白形式分析工具的特异性和敏感性缺陷，我们引入了一种混合微流体平台（DropBlot），该平台设计用于化学固定的单细胞中的蛋白质形式分析。DropBlot连续集成了基于液滴的封装和单个固定细胞的裂解，基于芯片微孔的抗原检索，靶抗原的单细胞免疫印迹。油包水液滴配方可以承受苛刻的化学品(SDS，6M尿素）和热条件（98°C，1-2小时)需要有效的抗原回收，并支持通过单细胞电泳对检索到的蛋白质靶标进行分析。我们展示了从不固定的蛋白质靶标检索，多聚甲醛固定（PFA），和甲醇固定的细胞。关键蛋白质靶标（HER2，GAPDH，EpCAM,从PFA固定的细胞中提取的波形蛋白）被解析并具有免疫反应性。与生物储存库相关，从存档了六年的人类乳腺肿瘤标本中检索到的DropBlot异形靶标，为保留生物样本的单细胞蛋白质生物标志物分析提供了工作流程。
