Electroosmotic Flow-Based Nanoinjection Technique Using a Nanopipette for Green Microalgae

Microalgae are gaining attention as promising candidates for CO₂ fixation and biomaterial production due to their non-competition with food and feed resources, as well as their high growth rates and photosynthetic efficiency. To expand their application as hosts for biomaterial production, it is essential to develop efficient and versatile gene modification technologies for microalgal metabolic engineering. Achieving this requires the establishment of an effective and broadly applicable material delivery method across diverse microalgal species. In this study, we developed a novel nanoinjection technique for single microalgal cells, utilizing electroosmotic flow through a nano-sized pipette (nanopipette). This nanopipette enables precise, automated delivery of solutions into cells at the femtoliter scale. Optimum injection conditions, including cell morphology, injection voltage, and injection time, were identified using fluorescein isothiocyanate (FITC)-labeled dextran in two green microalgae species, Haematococcus sp. and Tetraselmis sp. The method achieved injection efficiencies of 44% for Haematococcus sp. and 45% for Tetraselmis sp. This technique demonstrates substantial potential for efficient genome editing and subsequent metabolic engineering in a wide range of microalgae species.