Electrokinetically controlled fluid injection into unicellular microalgae

Abstract

Electrokinetically controlled microinjection is reported as an effective transport mechanism for microinjection into the wild-type strain of the widely studied model microalga Chlamydomonas reinhardtii. A microinjection system using glass capillary pipettes was developed to capture and impale the motile cells. To apply an electric field and induce electrokinetic flow (e.g., electrophoresis and electroosmosis), an electrode was inserted directly into the solution inside the impaling injection pipette and another electrode was inserted into the external cell media. The viability of the impaled cells was confirmed for more than an hour under 0.01 V using the fluorescein diacetate/propidium iodide dual fluorescent dye based assay. The viability was also found to increase almost logarithmically with decreasing voltage and to depend strongly on the solution within the injection pipette. Successful electrokinetic microinjection into cells was confirmed by both an increase in cell volume under an applied voltage and electric field dependent delivery of fluorescent fluorescein molecules into an impaled cell. Our study offers novel opportunities for quantitative delivery of biomolecules into microalgae and advancing the research and development of these organisms as biosynthetic factories.

Template‐Synthesized Vertical Needle Array as Injection Platform for Microalgae

“…For C. reinhardtii cells, immediate cell rupture upon impalement have been reported for needles with tips that are larger than 900 nm . We have also found that complete skewering of the C. reinhardtii induce immediate cell death, and when the needle density is too high ( e. g . 10 8 needles per cm 2 ), the cells rest on top of the needles (without being impaled) due to the “bed of nails” effect .…”

“…We have shown through similar assays that CC124 cells retain viability after impalement and injection by a hollow glass‐capillary needle . However, further studies are needed to assess the impact of hollow Pt/Ni NNA impalement on cell viability.…”

“…As the studies advance, hollow NNAs may offer more advantages over the solid NNAs for its ability to achieve time‐ and spatially‐resolved delivery, and to apply electrokinetic forces to quantitatively control delivery . The metallic template‐synthesized NNAs may also be used directly as electrodes that take advantage of electric‐field based manipulations: e. g .…”

Current Status and Perspective on the Use of Viral-Based Vectors in Eukaryotic Microalgae

“…Other methods include stable nuclear transformation systems for Pleurochrysis carterae using polyethylene glycol (PEG)-mediated transfer of hygromycin B-resistance genes [ 85 ]. Recent reports present genetic transformation of microalgae by bacterial conjugation [ 86 , 87 ] and gene injection [ 88 ]. In addition to these techniques, other emerging methods, such as cell-penetrating peptides, nanoparticles, metal–organic frameworks, and liposomes, have not yet been demonstrated in microalgae [ 12 , 89 , 90 , 91 ].…”

Controllable Fabrication of Small-Size Holding Pipets for the Nondestructive Manipulation of Suspended Living Single Cells

“…To achieve subsequent mechanical intracellular delivery to captured single cells, , negative pressure is generally combined with a microfluidic chip to trap single cells. ,, However, this method suffers from randomness in the intracellular delivery location after cell capture. A micropipet-based method was used to capture small-size suspended single cells of plants and carry out intracellular delivery, in which the holding pipet used has a sharp and thin-wall tip opening . Plant cells have cell walls, while animal cells do not.…”