MoNa – A Cost-Efficient, Portable System for the Nanoinjection of Living Cells

Abstract

Injection techniques to deliver macromolecules to cells such as microinjection have been around for decades with applications ranging from probing whole organisms to the injection of fluorescent molecules into single cells. A similar technique that has raised recent interest is nanoinjection. The pipettes used here are much smaller and allow for the precise deposition of molecules into single cells via electrokinetics with minimal influence on the cells’ health. Unfortunately, the equipment utilized for nanoinjection originates from scanning ion conductance microscopy (SICM) and is therefore expensive and not portable, but usually fixed to a specific microscope setup. The level of precision that these systems achieve is much higher than what is needed for the more robust nanoinjection process. We present Mobile Nanoinjection (MoNa), a portable, cost-efficient and easy to build system for the injection of single cells. Sacrificing unnecessary sub-nanometer accuracy and low ion current noise levels, we were able to inject single living cells with high accuracy. We determined the noise of the MoNa system and investigated the injection conditions for 16 prominent fluorescent labels and fluorophores. Further, we performed proof of concepts by injection of ATTO655-Phalloidin and MitoTracker Deep Red to living human osteosarcoma (U2OS) cells and of living adult human inferior turbinate stem cells (ITSC’s) following neuronal differentiation with the MoNa system. We achieved significant cost reductions of the nanoinjection technology and gained full portability and compatibility to most optical microscopes.

The new era of high throughput nanoelectrochemistry

“…140 Similar techniques have been developed to deliver molecules inside single cells with high spatial resolution. [141][142][143][144][145] Despite these studies being linked to high-throughput downstream biological analysis, the number of collectable samples is still limited by the lack of automation. Mukherjee et al 146 developed a fully automated nanofountain probe electroporation system based on angular approach SICM.…”

“…The technique was used to deliver amyloid-β aggregates on the surface of single macrophages to investigate the cellular response triggered by the aggregates. Similarly, Simonis et al 141 developed a portable and easy-to-build system to allow nanoinjections in single cells without the need for bulky and custom-made equipment, which would limit its use to equipped laboratories and highly skilled operators.…”

Methods for protein delivery into cells: from current approaches to future perspectives

“…One strategy to minimize the cellular impact of the injection process is to reduce the size of the probe to the nanoscale ( Figure 1B), which has been shown to improve the cell viability after injection [24][25][26][27][28]. One such probe-based device is the nanopipette, which is essentially a scaled-down micropipette with a nanometer-scale pore, a nanopore, at its tip [28][29][30][31][32]. Nanopipettes can be easily fabricated from quartz capillaries with a laser puller and the pore size can be precisely adjusted within the 10-300 nm range.…”

“…SICM has been successfully used for the high-resolution (20 nm) topographical imaging of living cells in culture [33][34][35][36]. The resultant topographical map can be then used to position the nanopipette anywhere on the cell membrane and precisely insert the nanopipette tip into the cell [28,30,37]. Because the nanopipettes are fitted with electrodes, the delivery of molecules into cells can be triggered by the application of a voltage of suitable polarity while achieving a high cell survival rate compared with micropipettes [28][29][30]32].…”

“…The resultant topographical map can be then used to position the nanopipette anywhere on the cell membrane and precisely insert the nanopipette tip into the cell [28,30,37]. Because the nanopipettes are fitted with electrodes, the delivery of molecules into cells can be triggered by the application of a voltage of suitable polarity while achieving a high cell survival rate compared with micropipettes [28][29][30]32]. Antibodies have been delivered into cells using this nanopipette based technique, demonstrating that it can be used for proteins [30].…”