@misc{oai:ir.soken.ac.jp:00001692,
 author = {畠山, 明子 and ハタケヤマ, アキコ and HATAKEYAMA, Akiko},
 month = {2016-02-17},
 note = {The injection of substances such as protein and DNA to living cells is indispensable to 
analyze molecular functions and to elucidate biological reactions. Especially, to investigate the cell-cell interaction and the cell fate determination, it is necessary to deliver object substances to a particular cell surrounded by a lot of neighboring cells. Although several techniques to deliver substances to single cells had been developed, a more simple and convenient technique, which can be applied to crowded single cells with the typical size of ~20 µm, is required. We have developed a new electroporation technique to deliver substances into single cells on target by applying electric pulses. This method uses the nano-electrode modified from a commercially available conductive-silicon cantilever for the atomic force microscope (AFM). The procedure to
prepare nano-electrodes is in two steps; one is to coat the whole surface of the cantilevers to insulate the surface electrically, and the other is to remove the coated agent at a tip. 
　　At first, we coated the surface of cantilevers by an aminosilane, 3-aminopropyltriethoxysilane (APTES). We use two solvents, hydrated toluene and a 5% water contained ethanol to deposit APTES. To characterize the electrical insulation of the coated cantilevers, impedance was measured at AC electrical fields in a frequency range from 10<sup>2</sup> to 10<sup>5</sup> Hz as well as at DC. As a result, the impedance of the cantilever increased by one order, indicating that the APTES coating is effective to improve surface isolation. Next, the deposited layer was  removed only at the tip by scratching a glass surface with AFM. Because a bubble due to  electrolysis of water was observed only at a tip of the scratched cantilever, the conductive area was proved to be limited at the electrode-tip. With the electrode, we succeeded in confining the electric field to the tip to perforate cell membrane of single cells. 
　　　This technique was applied to inject to fluorescent dye, propidium iodide (PI), into single cells of <i>Spodoptera frugiperda</i> (Sf9) in typical diameter of 10-20 µm. PI was added to the PBS solution containing Sf9 cells and the nano-electrode was approached onto the cell membrane. 
Then a square pulse was applied. The optimal pulse duration was determined at the constant amplitude by monitoring the injection efficiency in different durations. By selecting appropriate pulse duration, we succeeded in injecting the dye into Sf9 cells with the efficiency as high as 100%. In conclusion, the nano-electrodes coated by APTES with removed the coating layer at the tip works well for the electroporation.　Next, to apply our electroporation technique to protein, rhodamine-labeled avidin was added to PBS solution containing Sf9 cells. The Injection efficiency reached 67% by selecting appropriate pulse conditions. This result demonstrates that our method is highly effective to inject rhodamine-labeled avidin to Sf9 cells,  although the efficiency is lower than the case of PI.  Next, to estimate the invasiveness of our method to Sf9 cells, the viability was measured. We found that, all cells were alive in 18 h (100% viability), indicating that our method is less invasive. 
　　　To investigate the effect of the pulse condition, we applied multiple -square pulses for injecting PI and rhodamine-labeled avidin. Injection efficiencies of PI were measured at different frequencies, DC, 10 kHz and 50 kHz. At each frequency, the injection efficiency reaches a maximum at a particular pulse duration. We found that the optimal pulse duration increases with increasing frequency. The efficiency is 100% for DC and decreases slightly with increasing frequency in the frequency range less than 50 kHz. The similar tendency was observed when rhodamine-labeled avidin was injected. These results suggest that in the case of Sf9 cells, the single square pulse with the optimal pulse duration is sufficient to obtain the best electroporation efficiency. 
　　　Furthermore, we applied this technique to 16-cell-stage embryos of <i>Caenorhabditis elegans</i> with hard eggshells. The embryos were fixed to the glass bottom of a culture dish coated with poly-L-lysine. Like the case of Sf9 cells, the nano-electrode was approached vertically to an embryo to apply electric pulses. We succeeded the injection of PI into cells inside the shell by applying electric pulses with relatively large amplitude (13 V). By selecting the appropriate extracellular solution, the embryos repeated cell divisions after the PI injection until later stage in development. 
　　　In conclusion, our method is powerful enough to deliver substances into single cells surrounded by neighboring cells and promising to analyze biological reactions such as developmental mechanisms and cell-cell interaction., 総研大甲第1346号},
 title = {Electric introduction of protein into single cells with nano-electrodes},
 year = {}
}