7. Prior to each experiment, the chip is treated with oxygen
plasma, soaked in 1 mM PEG in ethyl alcohol at room temperature for 30 min, and then baked at 110
C for another 30 min.
This treatment coats the surface with a layer of PEG molecules
(see Note 4).
8. Planar electrode (as the chip assembly substrate and bottom
electrode) is comprised of a glass slide coated using chemical
vapor deposition with 100 nm Au.
9. A PDMS spacer on the surface of the planar electrode holds a
solution of the molecules to be delivered into the cells.
10. The fabricated chip is placed on top of the PDMS spacer with
the nanopores upward.
11. After cells are loaded on the chip and aligned using the magnetic tweezers, the top electrode is placed into the buffer
solution of the cells for electroporation.
12. To attach magnetic beads to the cells, depletion cocktail containing tetrameric antibody complexes targeting CD45 antigens (which is expressed on all cells used in this study) is mixed
with cells in PBS and allowed to incubate at room temperature
with regular mixing (see Note 5).
13. Dextran-coated magnetic microbeads are then added to the
cells in solution and once again incubated with mixing.
14. The cells are washed while using a magnet to remove any
unlabeled cells.
15. Cells are released by adding enough 0.05% trypsin to the dish
containing the adherent cells to cover a thin (~1 mm) layer of
solution.
16. Incubate cells for 2 min and agitate the dish to remove the cells
from the surface.
17. Count and remove enough cells to deposit them onto the
assembled device at a concentration equal to or less than
40,000 cells/cm
2 .
18. Programmable routines are used that rotate H XY and reverse
H Z to transport the labeled cells to electroporation sites (see
Note 6).
19. The electroporation is immediately turned on to transfect the
cells.
3.3 DEP
1. The cells are resuspended using trypsin and centrifugation, and
then they are placed in a low conductivity isotonic buffer
solution.
2. Using a prefabricated silicon nanopore chip and assembled, the
cells are loaded on the chip.
3D Nanochannel Array for High-Throughput Cell Manipulation and Electroporation
37
plasma, soaked in 1 mM PEG in ethyl alcohol at room temperature for 30 min, and then baked at 110
C for another 30 min.
This treatment coats the surface with a layer of PEG molecules
(see Note 4).
8. Planar electrode (as the chip assembly substrate and bottom
electrode) is comprised of a glass slide coated using chemical
vapor deposition with 100 nm Au.
9. A PDMS spacer on the surface of the planar electrode holds a
solution of the molecules to be delivered into the cells.
10. The fabricated chip is placed on top of the PDMS spacer with
the nanopores upward.
11. After cells are loaded on the chip and aligned using the magnetic tweezers, the top electrode is placed into the buffer
solution of the cells for electroporation.
12. To attach magnetic beads to the cells, depletion cocktail containing tetrameric antibody complexes targeting CD45 antigens (which is expressed on all cells used in this study) is mixed
with cells in PBS and allowed to incubate at room temperature
with regular mixing (see Note 5).
13. Dextran-coated magnetic microbeads are then added to the
cells in solution and once again incubated with mixing.
14. The cells are washed while using a magnet to remove any
unlabeled cells.
15. Cells are released by adding enough 0.05% trypsin to the dish
containing the adherent cells to cover a thin (~1 mm) layer of
solution.
16. Incubate cells for 2 min and agitate the dish to remove the cells
from the surface.
17. Count and remove enough cells to deposit them onto the
assembled device at a concentration equal to or less than
40,000 cells/cm
2 .
18. Programmable routines are used that rotate H XY and reverse
H Z to transport the labeled cells to electroporation sites (see
Note 6).
19. The electroporation is immediately turned on to transfect the
cells.
3.3 DEP
1. The cells are resuspended using trypsin and centrifugation, and
then they are placed in a low conductivity isotonic buffer
solution.
2. Using a prefabricated silicon nanopore chip and assembled, the
cells are loaded on the chip.
3D Nanochannel Array for High-Throughput Cell Manipulation and Electroporation
37