High-Throughput Microfluidic Electroporation (HTME): A Scalable, 384-Well Platform for Multiplexed Cell Engineering

Abstract

Electroporation-mediated gene delivery is a cornerstone of synthetic biology, offering several advantages over other methods: higher efficiencies, broader applicability, and simpler sample preparation. Yet, electroporation protocols are often challenging to integrate into highly multiplexed workflows, owing to limitations in their scalability and tunability. These challenges ultimately increase the time and cost per transformation. As a result, rapidly screening genetic libraries, exploring combinatorial designs, or optimizing electroporation parameters requires extensive iterations, consuming large quantities of expensive custom-made DNA and cell lines or primary cells. To address these limitations, we have developed a High-Throughput Microfluidic Electroporation (HTME) platform that includes a 384-well electroporation plate (E-Plate) and control electronics capable of rapidly electroporating all wells in under a minute with individual control of each well. Fabricated using scalable and cost-effective printed-circuit-board (PCB) technology, the E-Plate significantly reduces consumable costs and reagent consumption by operating on nano to microliter volumes. Furthermore, individually addressable wells facilitate rapid exploration of large sets of experimental conditions to optimize electroporation for different cell types and plasmid concentrations/types. Use of the standard 384-well footprint makes the platform easily integrable into automated workflows, thereby enabling end-to-end automation. We demonstrate transformation of E. coli with pUC19 to validate the HTME's core functionality, achieving at least a single colony forming unit in more than 99% of wells and confirming the platform's ability to rapidly perform hundreds of electroporations with customizable conditions. This work highlights the HTME's potential to significantly accelerate synthetic biology Design-Build-Test-Learn (DBTL) cycles by mitigating the transformation/transfection bottleneck.

Keywords: automation; electroporation; high-throughput; microfluidic; self-driving lab; strain engineering; synthetic biology; transfection; transformation.

Figure 1

(A) Photograph of an E-Plate. (B) Rendering of the interdigitated electrode geometry illustrating the outward capillary flow. (C) A 2 μL droplet of E. coli beads on the well surface without a surface tension trap. (D) A 2 μL droplet of E. coli in a well with a surface tension trap exhibits nearly complete wetting. (E) A 3 μL droplet of E. coli demonstrates complete wetting within the surface tension trap. Note that food dye was added to the cells to improve image contrast and visualization in (D,E). Note, the scale bar on A represents 10 mm and the scale bars on (B–E) represent 1 mm.

Figure 2

The HTME housing, containing all the mechanical and electrical components of the device. Key components are labeled, including the stepper motor, gearbox, spring loaded E-Plate clamping mechanism, Opentrons Peltier module (Long Island City, NY, USA), pulse generator and routing PCBs, E-Plate, and interface PCB.

Figure 3

Representative exponential pulse generator schematics for (A) typical cuvette electroporators and (B) the HTME.

Figure 4

Image of E. coli colonies expressing the selectable marker on a QTray (inner well dimensions: 3.0 × 2.5 cm) following successful transformation using the HTME platform. All wells were subjected to identical conditions with pulse parameters of 180 V and 5 ms time constant, using 0.02 ng/μL pUC19 plasmid. Note, a fraction of the recovered cells were plated to facilitate colony counting and prevent lawn formation (Section 2.5). The image is cropped from a QPix-generated composite of individually imaged wells. The full QTray image is available in the Supplementary Material.

Figure 5

Electric field distribution within 3.0 µL droplets with an applied voltage of 180 V shown as 2D cross-sectional views simulated using COMSOL Multiphysics V5.5. (A) A beaded hemispherical droplet and (B) a droplet in the surface tension trap. Both droplets have approximately 25% of their volume exposed to >10% of the maximum field strength of 18 kV/cm (colored region). The trap ensures consistent electrode coverage across wells. Future iterations of the trap geometry could enable lower droplet volumes achieving higher transformation efficiency while maintaining uniform droplet/electrode coverage. The color scale represents electric field strength in kV/cm. Note, this simulation utilizes water, with a relative permittivity of 80 and conductivity of 5.6 × 10⁻⁴ [S/m], as a representative model for illustrative purposes and does not account for the complex dielectric properties of cells and DNA.

Figure 6

(A) Normalized transformation efficiency from combined experimental runs indicates that transformation efficiency increases with increasing voltage and (B) normalized CV in CFU from combined experimental runs indicate that CFU variability decreases with increasing pulse voltage and plasmid concentration. 24 replicates were used per condition with a couple exceptions. A dispensing error occurred in the second run that caused all droplets for the 225 V, 0.16 ng/μL and half of the droplets for the 180 V, 0.16 ng/μL conditions to partially fill the E-Plate. As a result, the data for the 225 V, 0.16 ng/μL condition could not be combined and normalized. Instead, we employed a proportional ratio-based estimation approach to calculate the relative efficiency and CV for this condition as described in the Supplementary Material.