Fabrication and Performance Capabilities of a Flow-Through Microfluidic Device for Single-cell Electroporation

Introduction: Electroporation has risen as an alternative to viral-mediated gene delivery because, despite their high efficiency, viral vectors have considerable disadvantages: they are complex and expensive to manufacture, have limited payload capacity and rise safety concerns. However, challenges such as Joule heating in macroscale systems and low throughput in microscale systems are being addressed. Flow-through microfluidic systems offer potential solutions, increasing throughput and efficiency for clinical applications.
We have developed a flow-through electroporation system that utilizes a triple-layer laminar flow design to not only ensure precise cell positioning within the channel, but also single-file line flow. Additionally, this design minimizes cell exposure to localized electric field concentrations and potential cytotoxic electrochemical byproducts. We have characterized electro-transfection efficiency (eTE) and throughput for different pulsing protocols and electroporation buffers.

Materials and

Methods: The microfluidic system consists of a glass substrate with co-planar titanium/platinum electrodes and a polydimethylsiloxane (PDMS) microchannel. It includes a three-inlet one-outlet channel (3.9 cm x 300 μm x 45 μm) for hydrodynamic focusing to isolate individual cells. A master mold was created via soft lithography using an SU-8 photoresist pattern. A 10:1 PDMS mixture was cast onto this mold and cured to form a negative replica. Electrodes (125 μm wide with 185 μm separation) were made by defining patterns on glass, etching recesses, and depositing titanium/platinum layers through physical vapor deposition. PDMS channel and electrode-patterned glass were aligned and bonded using oxygen plasma treatment, creating a sealed microfluidic device. Copper wires were soldered to electrodes for electrical connectivity.
HEK-293 cells were suspended in electroporation buffer along with DNA plasmid encoding GFP. Sample and sheath fluid flow rates were adjusted for hydrodynamic focusing.
The electric fields were stimulated by unipolar square pulse trains characterized by a pulse amplitude, a pulse duration and a pulse frequency. As a cell passes between an electrode set, the applied field is controlled by the electrodes’ bias and spacing, while the pulse length is determined by the cell velocity and electrode width.
Twenty-four hours post-electroporation, GFP-expressing cells were counted in five randomly selected fields using the ImageJ software while employing fluorescence imaging. Electro-transfection efficiency (eTE) was calculated for each pulsing condition by dividing GFP-expressing cell count by viable cell count.

Results, Conclusions, and Discussions: This system was able to successfully electroporate single cells in a flow-through fashion with a maximum eTE equal to 62.34% at a throughput of 223 cells/sec utilizing a 100 uS/cm buffer. Increasing the frequency of the applied square pulses from 1kHz to 10kHz allowed higher amplitude pulses. eTE increased with the number of applied pulses and pulse amplitude. Two different electroporation buffers were used and tested. Although in past work from this lab, we had found that a higher conductivity buffer of 500 uS/cm yielded high eTE and high viability, within this high throughput device we found that the higher conductivity buffer was prone to induce electrolysis and hindered the ability of delivering pulses capable of electroporating the cells. Lastly, we assessed more complex pulsing patterns by applying weaker but longer pulses followed by strong but shorter pulses which yielded a significant increase in the eTE. To illustrate this, when 337 12.3 V pulses with a 200us duration were applied the cells in addition to 112 35V pulses with 10us duration, the eTE increased to 60%, compared to 28%, when just the 112 35V pulses with 10us duration were applied.
This study helps define that parameter space for high-throughput, continuous flow electroporation with high efficiency and can be integrated to enhance other microfluidic electroporation systems and capabilities, such as cell sensing and feedback control.

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