Silica-Based Nanopipettes for Rapid Living Single-Cell Transfection

Abstract

Intracellular delivery of materials with nanopipettes has become a critical method in gene editing and cell-based therapy because of its nanometer-scaled features capable of penetrating the cell membrane and targeting specific subcellular compartments—directing materials to the endoplasmic reticulum, nucleus, or mitochondria, for example. The geometrical parameters of nanopipettes directly affect delivery efficiency. However, the current methods for fabricating nanopipettes have the disadvantages of poor controllability, high cost, and difficult operation. To overcome these issues, we propose a real-time visualization method for fabricating nanopipettes based on pressured electrolyte chamber based wet etching to precisely determine the tip size during the fabrication process. A standard curve is plotted to provide a direction for fabrication, and tip inner diameters smaller than 10 nm can be controllably achieved. Furthermore, the intranuclear injection of proteins to living single cells (diameter < 30 μm) with a high spatial resolution is realized. And single-cell transfection through the intracellular delivery of plasmid based on a self-built living single-cell workstation is completed. This technique is expected to be used in the treatment of diseases, for high-resolution localization of organelles in living single cells without fluorescent labeling, and for subcellular omics analysis by mass spectrometry.