Microchip Electroporation

Tao Geng and Chang Lu*
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99354, USA.
*Department of Chemical Engineering, Virginia Tech, Blacksburg, VA 24061, USA.

Electroporation, also termed as electropermeabilization, is a robust physical method for breaking cell membranes to either release intracellular components out of cells or introduce exogenous molecules into cells.1-3 When an electric field is applied to a cell, cell membrane charges like a capacitor, as it has several orders of magnitude higher conductivity than cytoplasm and extracellular medium. Thus, a potential difference is formed across the intact cell membrane within an extremely short charging time (on the scale of microseconds). As the transmembrane potential (ΔψE) exceeds a critical threshold (typically 0.2 to 1 V irrespective of cell types), electroporation occurs with nanoscale eletropores created within the plasma membrane via the localized structural rearrangements of lipid molecules.1,2 Assuming that the cell is a sphere and the membrane charging time is much shorter than the electric field duration, the ΔψE induced by an external electric field can be described using the following equation2,4:

ΔψE=0.75 g(λ)aE cosθ(1)

where g(λ) is a function of the conductivities of cytoplasm, cell membrane and extracellular buffer, a is the cell diameter, E is the electric field intensity, and θ is the angle between the direction of the electric field E and the normal from the cell center to a point on the membrane surface.

Depending on the degree of the conformational changes in the membrane structure, electroporation can be reversible or irreversible. If the electric pulse amplitude/duration is not too high/long, the pores are transient and can rapidly reseal after the removal of the field, ensuring that the cells remain viable. Reversible electroporation has been widely applied to the delivery of diverse foreign molecules into cells, including small-molecule fluorescent dyes, drugs, oligonucleotides, DNA, RNA, peptides, proteins, and nanoparticles etc.5-8 When the electric pulses are highly intensive, excessive electroporation leads to the generation of permanent pores and cell death, followed by rapid release of intracellular molecules due to electrophoretic forces. Irreversible electroporation has been used to selectively kill target cells for cell enrichment9,10, inactivation of microorganisms in food products11, and release intracellular materials (e.g., DNA, RNA, enzymes, and other cytoplasmic proteins) for cell analysis or the production of therapeutic proteins12-14.

Conventional electroporation is implemented by exerting short electric pulses of defined duration and intensity to a cuvette with electrodes fabricated out of aluminum, stainless steel, platinum or graphite, and arranged in a plate-to-plate manner.15 A pulse generator such as special capacitor discharge equipment is required to generate high-voltage pulses. By tuning the electric parameters (e.g., electric pulse amplitude, duration, frequency and shape), electroporation efficiency and cell viability (specially for molecular delivery) can be optimized.16 Although traditional electroporation systems have been widely used, they require high voltage input and suffer from adverse environmental conditions such as electric field distortion, local pH variation, metal ion dissolution and excess heat generation, leading to low electroporation efficiency.

Due to its physical nature, electroporation can be easily miniaturized on microfluidic platforms. Microfluidic electroporation systems overcome many drawbacks of bench-scale electroporators owing to its unique characteristics of miniaturization and integration.17-19 First, microfluidic electroporation devices are fabricated with standard microfabrication techniques, and hence a wide spectrum of microelectrodes can be incorporated into the microchips to generate the field necessary for electroporation. By shrinking the distance between microelectrodes to a few tens of micrometers20-22 or creating physical constraints with subcellular dimensions23-25, the required voltage is dramatically decreased to a few volts. Second, the electroporation microchips provide relatively more uniform electric field distribution, favorable fluidic and chemical environment, and rapid heat dissipation in small-volume microstructures. Third, individual cells could be manipulated on chips to probe single-cell dynamics and identify cell heterogeneity. The miniaturization of the systems also makes them very suitable for assays involving rare cell populations and expensive reagents due to the substantially reduced sample consumption. Fourth, the flow-through format of the microfluidic electroporation also allows the continuous processing of large cell populations with high throughput to obtain statistically meaningful results at the single-cell level or produce in a large volume.23,25-29 Fifth, the utilization of transparent materials (e.g., polydimethylsiloxane and glass) for microchips allows in situ observation and real-time monitoring of electroporation process using fluorescent probes, which facilitates the exploration of electroporation mechanism. Finally, microfluidic electroporation can be integrated with other analytical processes such as dielectrophoresis, electrophoresis, electroosmosis and hybridization to implement a total analysis analytical system, which is especially critical to of intracellular content analysis.30-44 Based on the strategies used to facilitate electroporation at the microscale, microfluidic electroporation methods are divided into five categories: (1) electrode incorporation and configuration, (2) channel geometry variation and constriction structures, (3) hydrodynamics-enhanced electroporation, (4) compartmentalized electroporation, and (5) other miscellaneous methods. Figure 1 demonstrates representative microfluidic electroporation methods.

Figure 1. Schematic of microfluidic electroporation methods. (a) Electrode incorporation and configuration: interdigitated 2D planar electrodes incorporated in a microchamber; (b) Channel geometry variation and constriction structures: a fluidic channel comprising a number of alternating wide and narrow sections where cells undergo electroporation while flowing through the narrow section; (c) Hydrodynamics-enhanced electroporation: hydrodynamic focusing for single-cell electroporation; (d) Compartmentalized electroporation: single-cell electroporation within a microfluidic droplet surrounded by oil.

The most straightforward way to implement electroporation at the microscale is constructing microelectrode structures inside simple microchannels or microchambers. The design of the microelectrodes is therefore of crucial importance for the electroporation process, because the geometry defines the electric field distribution and uniformity and hence greatly affects the electroporation efficiency. The electrodes utilized in microfluidic electroporation can be generally sorted into four groups: (1) parallel plate electrodes, (2) coplanar electrodes, (3) 3D electrodes, and (4) wire electrodes. Parallel plate electrodes that mimic the architecture of commercial electroporation systems is one of the simplest arrangements.45-54 In such systems, the microchannel is sandwiched between two substrates coated with various electrodes such as gold, aluminum, stainless steel and indium tin oxide, and therefore the electric field is uniformly distributed in between the space of the two electrodes. The metal electrodes could also be modified with high-aspect-ratio nanoscale structures to locally focus electric field intensity to the surface of the cell membranes.48,49,51,55 Cell suspension can be continuously flowed through the channel, but cells cannot be visualized in real-time when metal electrodes are involved. Microelectrodes are also often arranged in coplanar configuration due to the convenience in the microfabrication and visualization. However, the electric field distribution is more complex in such architecture, and hence a variety of electrode geometries have been proposed to obtain optimal electric fields for electroporation. The microelectrode geometries include parallel strip electrodes56-62, interdigitated electrodes in rectangular33-35,63-70, castellated37,44, circular38-40, curved71 or saw-tooth32,42,43 formats, and circular/square electrode arrays30,31,72-77. Combined with microchannel or microchamber structures, the systems could process either suspended or adherent cells. However, 2D planar electrodes suffer from non-uniform electric field distribution and high possibility of decaying away from the metal layers due to water electrolysis. Therefore, 3D electrodes including vertical sidewall electrodes36,78-84, electrode arrays in the form of pillars85,86, needles87,88 or nails89,90, and “liquid electrodes”91-94 have been reported to address these challenges in spite of the complexity in manufacturing. Although microelectrodes offers diverse forms of electric fields on-chip, it is still beneficial to use macroscale wire electrodes by simply inserting them in the open reservoirs connected to the microchannel network. Platinum wires and Ag/AgCl wires are two most commonly used electrodes because of their chemical stability.9,95-122

Constriction segments or structures in microchannels or microchambers can generate high localized electric fields, and thus electroporation occurs when cells of interest are flow through or positioned within such constricted regions. Due to the high electrical resistance of the small constriction regions, cells experience high field intensity in such configurations even when the overall input voltage is low. A simple fluidic channel comprising a number of alternating wide and narrow sections has been created for flow-through electroporation based on a constant voltage.9,103-106,108,109,112-114,116-120 Appropriate combination of the voltage and the channel geometry yields high field intensity in the narrow sections that is beyond the electroporation threshold and low field intensity in the wide sections that does not affect membrane integrity. Cells undergo electroporation while flowing through the narrow section(s), and the duration of electroporation is determined by the residence time(s) in the narrow section(s). Having multiple narrow sections in the channel renders flowing cells subject to field variations equivalent to multiple pulses. Alternatively, the constriction segments of a channel could also be constructed by reducing the depth of a segment of the channel.123,124 Moreover, microscale holes and channels with subcellular dimensions have been used for the entrapment and electroporation of single or multiple cells.27,125-136 More recently, nanoscale structures such as nanopores, nanostraws, and nanochannels have been implemented to provide even smaller constriction features and offer higher precision and efficiency for electroporation.137-144

Microfluidics creates favorable hydrodynamic conditions for cell manipulation in fluidic networks, which has been harnessed to enhance the performance of microfluidic electroporation.115,119,145-149 For instance, a spiral-shaped electroporation channel enabled the system to generate permeabilization over the whole cell membrane surface, thereby substantially enhancing the electroporation efficiency without compromising the cell viability.115 Also, hydrodynamic focusing was incorporated to facilitate single cell electroporation.119,145

Confining cells into tiny reaction volumes offers a number of advantages for electroporation including increased contact between the cell and the delivered molecule as well as potential for single cell/molecule screening and analysis. Microfluidics provides an ideal platform for the spatiotemporally regulated electroporation processes in the form of either droplets60,150-154 or microwell arrays31,59,69,71,155-157. These formats are especially suitable for single-cell electroporation.

Other than the abovementioned technologies, miscellaneous electroporation methods have been developed on chips based on microvalves107, salt bridges158 and light29,159,160.

In conclusion, the fusion of microfluidics with electroporation has created a myriad of versatile tools for analyzing intracellular contents and delivering foreign molecules into cells at microscale. Microfluidic electroporation allows more precise control over the process and more flexible integration with other tools than their bench-scale counterpart. Additionally, microfluidic platform provides unique opportunities for incorporating hydrodynamic effects into electroporation-based processes, and these effects often produce results that are otherwise not achievable. These exciting breakthroughs will benefit biotechnology and life science in broad realm including cell therapy, electrochemotherapy, large-scale functional screening of genome, and cell biology studies.

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