Nanofountain Probe Electroporation Enables Versatile Single‐Cell Intracellular Delivery and Investigation of Postpulse Electropore Dynamics

Abstract

Introducing exogenous molecules into cells with high efficiency and dosage control is a crucial step in basic research as well as clinical applications. Here, the capability of the nanofountain probe electroporation (NFP‐E) system to deliver proteins and plasmids in a variety of continuous and primary cell types with appropriate dosage control is reported. It is shown that the NFP‐E can achieve fine control over the relative expression of two cotransfected plasmids. Finally, the dynamics of electropore closure after the pulsing ends with the NFP‐E is investigated. Localized electroporation has recently been utilized to demonstrate the converse process of delivery (sampling), in which a small volume of the cytosol is retrieved during electroporation without causing cell lysis. Single‐cell temporal sampling confers the benefit of monitoring the same cell over time and can provide valuable insights into the mechanisms underlying processes such as stem cell differentiation and disease progression. NFP‐E parameters that maximize the membrane resealing time, which is essential for increasing the sampled volume and in meeting the challenge of monitoring low copy number biomarkers, are identified. Its application in CRISPR/Cas9 gene editing, stem cell reprogramming, and single‐cell sampling studies is envisioned.

Deep Learning‐Assisted Automated Single Cell Electroporation Platform for Effective Genetic Manipulation of Hard‐to‐Transfect Cells

“…We have shown in a previous study that sufficient contact is made in this process to increase the transmembrane potential beyond the electroporation threshold. [ 47 ] We have also investigated the effect of the extent of contact on delivery efficiency and cell viability. [ 41 ] In the current study, contact is determined when the resistance change (≈300–500 kΩ) exceeds 1% of the baseline (≈30–50 MΩ).…”

Nanostraw‐Assisted Cellular Injection of Fluorescent Nanodiamonds via Direct Membrane Opening

“…[ 46,47 ] Similarly, the resealing of the membrane is much slower in the present case (30–60 min), than what has been shown in the case of nanofountain probe electroporation (few tens of seconds). [ 48 ] We can possibly attribute these differences to longer electroporation pulses and a higher number of pulses, as it has been shown that both the membrane pore size and the resealing time increases with the number of pulses and pulse duration. [ 49 ] Increase in the local electrical field and increased membrane tension on nanostraws may also be an explanation.…”

“Writing biochips”: high-resolution droplet-to-droplet manufacturing of analytical platforms

“…This system has been recently used to deliver proteins and plasmids into cells, also providing precise intracellular sampling of model fluorescent proteins, such as calcein and tdTomato. 89 The authors used BSA-AF488 protein as a model biomolecular system to deliver into single cells, in particular analyzing the effect of the number of voltage pulses (typically around 1 V) in enabling functional membrane opening. Excellent plasmid transfection efficiency in HEK 293FT cells was achieved, outclassing the Lipofectamine based assay.…”