Continuous microfluidic flow-through protocol for selective and image-activated electroporation of single cells

Electroporation of cells is a widely-used tool to transport molecules such as proteins or nucleic acids into cells or to extract cellular material. However, bulk methods for electroporation do not offer the possibility to selectively porate subpopulations or single cells in heterogeneous cell samples. To achieve this, either presorting or complex single-cell technologies are required currently. In this work, we present a microfluidic flow protocol for selective electroporation of predefined target cells identified in real-time by high-quality microscopic image analysis of fluorescence and transmitted light. While traveling through the microchannel, the cells are focused by dielectrophoretic forces into the microscopic detection area, where they are classified based on image analysis techniques. Finally, the cells are forwarded to a poration electrode and only the target cells are pulsed. By processing a heterogenically stained cell sample, we were able to selectively porate only target cells (green-fluorescent) while non-target cells (blue-fluorescent) remained unaffected. We achieved highly selective poration with >90% specificity at average poration rates of >50% and throughputs of up to 7200 cells per hour.