The First Affiliated Hospital of Sun Yat-Sen University; State Key Laboratory of Optoelectronic Materials and Technologies, School of Electronics and Information Technology , Sun Yat-Sen University , Guangzhou 510006 , China.
Pritzker School of Medicine , University of Chicago , Chicago , Illinois 60637 , United States.
ACS Sens. 2018 Sep 28;3(9):1675-1682. doi: 10.1021/acssensors.8b00367. Epub 2018 Sep 5.
Techniques used to understand the dynamic expression of intracellular proteins are critical in both fundamental biological research and biomedical engineering. Various methods for analyzing proteins have been developed, but these methods require the extraction of intracellular proteins from the cells resulting in cell lysis and subsequent protein purifications from the lysate, which limits the potential of repetitive extraction from the same set of viable cells to track dynamic intracellular protein expression. Therefore, it is crucial to develop novel methods that enable nondestructive and repeated extraction of intracellular proteins. This work reports a hollow nanoneedle-electroporation system for the repeated extraction of intracellular proteins from living cells. Hollow nanoneedles with ∼450 nm diameter were fabricated by a material deposition and etching process, followed by integration with a microfluidic device. Long-lasting electrical pulses were coupled with the nanoneedles to permeate the cell membrane, allowing intracellular contents to diffuse into the microfluidic channels located below the cells via hollow nanoneedles. Using lactate dehydrogenase B (LDHB) as the model intracellular protein, the nanoneedle-electroporation system effectively and repeatedly extracted LDHB from the same set of cells at different time points, followed by quantitative analysis of LDHB via standard enzyme-linked immunosorbent assay. Our work demonstrated an efficient method to nondestructively probe intracellular protein levels and monitor the dynamic protein expression, with great potential to help understanding cell behaviors and functions.
用于理解细胞内蛋白质动态表达的技术在基础生物学研究和生物医学工程中都至关重要。已经开发出了各种用于分析蛋白质的方法，但这些方法需要从细胞中提取细胞内蛋白质，导致细胞裂解，随后从裂解物中纯化蛋白质，这限制了从同一组活细胞中重复提取以跟踪细胞内蛋白质动态表达的潜力。因此，开发能够进行非破坏性和重复提取细胞内蛋白质的新方法至关重要。本工作报道了一种用于从活细胞中重复提取细胞内蛋白质的中空纳米针电穿孔系统。通过材料沉积和蚀刻工艺制造出直径约为 450nm 的中空纳米针，然后与微流控器件集成。将持久的电脉冲与纳米针耦合，使细胞膜穿孔，使细胞内的内容物通过中空纳米针扩散到位于细胞下方的微流道中。使用乳酸脱氢酶 B（LDHB）作为模型细胞内蛋白质，纳米针电穿孔系统有效地从同一组细胞在不同时间点重复提取 LDHB，并通过标准酶联免疫吸附测定法对 LDHB 进行定量分析。我们的工作展示了一种有效且非破坏性的探测细胞内蛋白质水平和监测动态蛋白质表达的方法，具有帮助理解细胞行为和功能的巨大潜力。
