Impedance analysis of adherent cells after in situ electroporation-mediated delivery of bioactive proteins, DNA and nanoparticles in \xb5L-volumes

Judith A Stolwijk,Joachim Wegener

doi:10.1038/s41598-020-78096-6

Abstract

Specific intracellular manipulation of animal cells is a persistent goal in experimental cell biology. Such manipulations allow precise and targeted interference with signaling cascades, metabolic pathways, or bi-molecular interactions for subsequent tracking of functional consequences. However, most biomolecules capable of molecular recognition are membrane impermeable. The ability to introduce these molecules into the cytoplasm and then to apply appropriate readouts to monitor the corresponding cell response could prove to be an important research tool. This study describes such an experimental approach combining in situ electroporation (ISE) as a means to efficiently deliver biomolecules to the cytoplasm with an impedance-based, time-resolved analysis of cell status using electric cell-substrate impedance sensing (ECIS). In this approach, gold-film electrodes, deposited on the bottom of regular culture dishes, are used for both electroporation and monitoring. The design of the electrode layout and measurement chamber allows working with sample volumes as small as 10 µL. A miniaturized setup for combined electroporation and impedance sensing (µISE-ECIS) was applied to load different adherent cells with bioactive macromolecules including enzymes, antibodies, nucleic acids and quantum dot nanoparticles. The cell response after loading the cytoplasm with RNase A or cytochrome c (in the presence or absence of caspase inhibitors) was tracked by non-invasive impedance readings in real-time.

Highlights

The transfer of nucleic acids including DNA, RNA, and siRNA into living mammalian cells has evolved as an important tool across all fields of biomedical research as these molecules enable studying or altering cellular functions[1]
The delivery of proteins and peptides including antibodies and enzymes or enzyme substrates to interfere with cell function on the protein level constitutes a long-sought goal in cell biology as the application of these molecules is otherwise limited to extracellular targets or fixed and permeabilized tissues[2,3]
A myriad of nanoparticle probes or sensors have been developed over the last decades that require precise targeting inside the cytoplasm for defined intracellular sensing[4]

Summary

Introduction

The transfer of nucleic acids including DNA, RNA, and siRNA into living mammalian cells has evolved as an important tool across all fields of biomedical research as these molecules enable studying or altering cellular functions[1]. Electrochemical approaches have been used to monitor membrane integrity of single cells during pulsing[8], the barrier function of cell layers before and after in situ electroporation[9], the effect of suspension electroporation on post-pulse cell layer formation[10] or the electrical properties of a tissue[11] Whereas these techniques were essentially applied to monitor the invasiveness of an electric pulse, none of them was used to record the cell response to the presence of bioactive molecules that had been delivered to the cytoplasm by means of pulsing. Can be a challenge for which different approaches have been proposed by other groups[15,16,17,18,19]

Methods

Results

Conclusion