Electron Microscopy
Cells were fixed overnight at 4oC using a fixative solution that comprises 25% of 0.4M cacodylate buffer, 12.5% of 16% formaldehyde, 10% of 25% glutaraldehyde and the rest being water. The cells are then washed thrice with water and dehydrated using increasing concentration of acetonitrile. The samples are then mounted on stubs using carbon tape and sputter-coated with 5nm of gold-palladium.
Electrical Stimulation
Various ES parameters in combination with chemical induction were studied. For ES, 5mm x 5mm array of nanofins were used after coating them with gold. ADSC cells were seeded on to of the nanofins at an initial seeding density of 30,000cells/cm2 with the help of a 10mm cloning cylinder. Parameters ranging from 50mV to 500mV were studied
Results
A. Nanopillars fabrication
Arrays of 5mm x 5mm were fabricated, as described in the experimental methods section. Optimized fabrication methodology allowed for tunable geometry with control over nanopillar density, tip diameter, height as shown in Table 1. The density of the pillars was controlled using appropriate mask patterning, while the height of the pillars was controlled in the RIE. The tip diameter was controlled within few tens of nanometers by an optimized combination of anisotropic and isotropic etch. The sharpness of tip is crucial to increase the probability of cell penetration.
Table 1. Variation in Nanoelectrode
| Nanopillar - 1 | Nanopillar/fin - 2 | Nanopillar/fin - 3 |
Tip dimension | ~100nm x 100nm | ~100nm x 3µm | ~100nm x7.5µm |
Height | ~5-6 µm | ~5-6 µm | ~5-6 µm |
Interpillar distance | ~11 x 11 µm | ~15 x 11 µm | ~19 x 11 µm |
Density | 8.3x103pillar/cm2 | 6.6x103pillar/cm2 | 4.8x103pillar/cm2 |
Table 1 describes the three different nanopillar or fin configurations. SEM images of the different configurations are shown in Fig. 2. The difference between the nanopillars and nanofins are that nanopillars have the same tip width and breadth while nanofins have different tip width and breadth. The advantage of the nanofin configuration over nanopillars is that nanofins have reduced interfacial impedance and reduced electrode resistance.