Single cell and neural process experimentation using laterally applied electrical fields between pairs of closely apposed microelectrodes with vertical sidewalls

Abstract

As biomedical research has moved increasingly towards experimentation on single cells and subcellular structures, there has been a need for microscale devices that can perform manipulation and stimulation at a correspondingly small scale. We propose a microelectrode array (MEA) featuring thickened microelectrodes with vertical sidewalls (VSW) to focus electrical fields horizontally on targets positioned in between paired electrodes. These microelectrodes were fabricated using gold electroplating that was molded by photolithographically patterned SU-8 photoresist. Finite element modeling showed that paired VSW electrodes produce more uniform electrical fields compared to conventional planar microelectrodes. Using paired microelectrodes, 3µm thick and spaced 10µm apart, we were able to perform local electroporation of individual axonal processes, as demonstrated by entry of EGTA to locally chelate intra-axonal calcium, quenching the fluorescence of a pre-loaded calcium indicator dye. The same electrode configuration was used to electroporate individual cells, resulting in the targeted transfection of a transgene expressing a cytoplasmically soluble green fluorescent protein (GFP). In addition to electropration, our electrode configuration was also capable of precisely targeted field stimulation on individual neurons, resulting in action potentials that could be tracked by optical means. With its ability to deliver well-characterized electrical fields and its versatility, our configuration of paired VSW electrodes may provide the basis for a new tool for high-throughput and high-content experimentation in broad areas of neuroscience and biomedical research.

Microfluidic electroporation for cellular analysis and delivery

“…To achieve single-cell resolution, the gaps between adjacent electrodes should be close enough to allow only one cell to be confined between them. By replacing the planar electrodes 54 , 3D vertical sidewall electrodes were used to improve the spatial uniformity of electric treatment on single cells 93, 95, 96 . Using this configuration, single neurons could be transfected with plasmid DNA, and in particular, local delivery of EGTA into a subcellular axon was also demonstrated.…”

Mapping of bionic array electric field focusing in plasmid DNA-based gene electrotransfer

“… 25 , 26 , 27 Closely apposed electrodes (for example, created by ion deposition and photolithography) with sub-millimetre spacing for electroporation of immediately adjacent cells or cell processes within culture chambers have also been investigated. 28 , 29 In such cases, the voltages required to achieve electroporation-based delivery of reporter gene constructs are considerably reduced over conventional electroporation modes (typically less than 10 V in comparison with several 100s V or even 1000 V in conventional electroporation).…”

Novel Aspects of Neuronal Differentiation In Vitro and Monitoring with Advanced Biosensor Tools

“…Electrical stimulation and directed local electroporation of concrete axons have been achieved by using a MEA in combination with thickened microelectrodes with vertical sidewalls separated by the appropriate distance to host an axon [72]. By combining patch clamp detection of intracellular membrane potential and the application of voltage pulses through the electrodes of a MEA, it could be observed that it is possible to controllably electroporate neurons with the underlying electrodes of the MEA, which could be very useful for transfection purposes.…”