Presentation Information
[4Marine-10]Single-Cell Nanoinjection Platform for Efficient Intracellular Delivery in Diverse Microalgae
○Makoto Mochizuki1, Daisuke Nojima1, Kaoruko Akasaka2, Tomoko Yoshino2, Tsuyoshi Tanaka2 (1. Yokogawa Electric Corporation (Japan), 2. Tokyo University of Agriculture and Technology (Japan))
Keywords:
Microalgae,Nanoinjection
[Purpose]
Microalgae are promising hosts for sustainable biomanufacturing because of their ability to fix CO2 and produce valuable compounds. However, genetic modification of microalgae remains difficult, especially in non-model species with rigid cell walls, because existing intracellular delivery methods are inefficient and poorly versatile. In this study, we aimed to develop a nanopipette-based single-cell nanoinjection platform utilizing electroosmotic flow and to evaluate whether this system enables efficient and precise intracellular delivery across different microalgal species through optimization of injection conditions and quantitative assessment of delivery efficiency.
[Method]
We developed a nanopipette-based nanoinjection system (Single Cellome Unit SU10) utilizing electroosmotic flow for deterministic intracellular delivery. The nanopipette, with a nanoscale tip diameter (approximately 30 nm), enables femtoliter-scale injection into individual cells with high spatial precision. The system automatically detects the cell surface based on ionic current changes, followed by controlled membrane penetration and injection. Key parameters, including applied voltage and injection duration, were systematically optimized using FITC-labeled dextran as a model biomolecule. The method was evaluated using representative green microalgae species, including Haematococcus sp. and Tetraselmis sp.
[Results & Consideration]
The developed nanoinjection system achieved efficient intracellular delivery in Haematococcus sp. and Tetraselmis sp., with optimized injection efficiencies of approximately 44–45%, which exceed those of conventional methods. Delivery efficiency depended on cell morphology, with successful injection observed in flagellated and palmelloid cells, while little to no delivery was achieved in intermediate and cyst cells, likely due to increased cell wall rigidity. The high injection efficiency is attributed to controlled single-cell targeting, which enables direct delivery into selected cells and eliminates the need for large-scale screening. In addition, precise control of injection volume through voltage and time modulation allows quantitative and reproducible delivery. Injected cells retained viability, indicating minimal cellular damage. These results demonstrate that the system provides a robust and broadly applicable platform for intracellular delivery in microalgae.
[Conclusion]
We have established a novel electroosmotic flow-based nanoinjection platform that enables efficient and precise intracellular delivery in diverse microalgae species. This method overcomes the limitations imposed by rigid cell walls and provides a powerful tool for advancing genetic engineering in both model and non-model microalgae. By shifting from random to controlled delivery, this platform holds significant potential for accelerating microalgal biotechnology and expanding its applications in next-generation sustainable biomanufacturing.
Microalgae are promising hosts for sustainable biomanufacturing because of their ability to fix CO2 and produce valuable compounds. However, genetic modification of microalgae remains difficult, especially in non-model species with rigid cell walls, because existing intracellular delivery methods are inefficient and poorly versatile. In this study, we aimed to develop a nanopipette-based single-cell nanoinjection platform utilizing electroosmotic flow and to evaluate whether this system enables efficient and precise intracellular delivery across different microalgal species through optimization of injection conditions and quantitative assessment of delivery efficiency.
[Method]
We developed a nanopipette-based nanoinjection system (Single Cellome Unit SU10) utilizing electroosmotic flow for deterministic intracellular delivery. The nanopipette, with a nanoscale tip diameter (approximately 30 nm), enables femtoliter-scale injection into individual cells with high spatial precision. The system automatically detects the cell surface based on ionic current changes, followed by controlled membrane penetration and injection. Key parameters, including applied voltage and injection duration, were systematically optimized using FITC-labeled dextran as a model biomolecule. The method was evaluated using representative green microalgae species, including Haematococcus sp. and Tetraselmis sp.
[Results & Consideration]
The developed nanoinjection system achieved efficient intracellular delivery in Haematococcus sp. and Tetraselmis sp., with optimized injection efficiencies of approximately 44–45%, which exceed those of conventional methods. Delivery efficiency depended on cell morphology, with successful injection observed in flagellated and palmelloid cells, while little to no delivery was achieved in intermediate and cyst cells, likely due to increased cell wall rigidity. The high injection efficiency is attributed to controlled single-cell targeting, which enables direct delivery into selected cells and eliminates the need for large-scale screening. In addition, precise control of injection volume through voltage and time modulation allows quantitative and reproducible delivery. Injected cells retained viability, indicating minimal cellular damage. These results demonstrate that the system provides a robust and broadly applicable platform for intracellular delivery in microalgae.
[Conclusion]
We have established a novel electroosmotic flow-based nanoinjection platform that enables efficient and precise intracellular delivery in diverse microalgae species. This method overcomes the limitations imposed by rigid cell walls and provides a powerful tool for advancing genetic engineering in both model and non-model microalgae. By shifting from random to controlled delivery, this platform holds significant potential for accelerating microalgal biotechnology and expanding its applications in next-generation sustainable biomanufacturing.
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