School of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.
School of Electronic and Electrical Engineering and Pollard Institute, University of Leeds, Leeds, LS2 9JT, UK.
Nat Commun. 2024 May 23;15(1):4403. doi: 10.1038/s41467-024-48608-3.
Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here we describe a platform that uses nanopipettes to deliver defined numbers of macromolecules into cultured cell lines and primary cells at single molecule resolution. In the nanoinjection platform, the nanopipette is used as both a scanning ion conductance microscope (SICM) probe and an injection probe. The SICM is used to position the nanopipette above the cell surface before the nanopipette is inserted into the cell into a defined location and to a predefined depth. We demonstrate that the nanoinjection platform enables the quantitative delivery of DNA, globular proteins, and protein fibrils into cells with single molecule resolution and that delivery results in a phenotypic change in the cell that depends on the identity of the molecules introduced. Using experiments and computational modeling, we also show that macromolecular crowding in the cell increases the signal-to-noise ratio for the detection of translocation events, thus the cell itself enhances the detection of the molecules delivered.
通过输送外源大分子来控制培养细胞的操作是实验生物学的基石。在这里，我们描述了一个使用纳米移液器以单分子分辨率将定义数量的大分子递送到细胞系和原代细胞中的平台。在纳米注射平台中，纳米移液器既用作扫描离子电导显微镜 (SICM) 探头，也用作注射探头。在将纳米移液器插入细胞到定义的位置和预定义的深度之前，SICM 用于将纳米移液器定位在细胞表面上方。我们证明，纳米注射平台能够以单分子分辨率定量地将 DNA、球形蛋白质和蛋白质原纤维递送到细胞中，并且递送到细胞中会导致细胞表型发生变化，这取决于引入的分子的特性。我们还通过实验和计算建模表明，细胞中的大分子拥挤会增加转位事件检测的信噪比，从而使细胞本身增强了对递送到细胞中的分子的检测。
