Patterned and switchable surfaces for biomaterial applications

Chapter 2 – Spatially controlled electro-stimulated DNA adsorption and desorption for biodeviceapplicationswater to ensure even DNA dispersion. Effectene Transfection Kit (Qiagen) was usedto enhance transfection efficiency. To prepare the transfection solution, 4 lenhancer was added to 37 l DNA condensation buffer (EC buffer) and incubated atroom temperature for 10 mins. 6 l effectene was then added and after vortexing, 0.5l of the complete transfection solution was dispensed over the bound DNA andallowed to air dry. Patterned ALAPP/PEG surfaces were arrayed with DNA using aPerkin Elmer Piezoarray. Typically 300 pl of solvent was dispensed per spot.pEGFP-N1 was spotted at a concentration of 0.1 mg/ml. Prepared transfectionreagent was diluted 4x and spotted on top of the DNA. Samples were then washedwith PBS, pH = 7.4 and placed in a custom-built transfection cell. The cell wasmachined from Teflon to produce a cube-shaped incubation chamber withdimensions of 2x2x2 cm 3 . Samples placed in the cell were clamped into position bystainless steel screws. Contact of the screws with the sample enabled the applicationof a voltage to the samples. HEK 293 cells were seeded at 1.0x10 5 cells/cm 2 to thetransfection cell and DMEM media was added to a total volume of 4 ml. After 4 hrincubation, -750 mV was applied for 0, 0.5, 1 and 5 min with the substrate as theworking electrode, an Ag/AgCl/saturated KCl reference electrode and a platinumauxiliary electrode. Cells were incubated for further 20 hr before being characterisedmicroscopically. HEK 293 cells were counterstained using the Hoechst 33342 dye(ex 350 nm, em 460 nm) (Molecular Probes), which is incorporated into the nucleusof all living cells, enabling the visualisation of the total cell population. Cells wereincubated in 10 g/ml Hoechst solution for 10 min, after which cells were washedwith PBS, pH = 7.4. Cells growing over DNA spots were visualised with a Leitzfluorescence microscope and images were captured using a Nikon Digital Sight DS-L1 and a Nikon Digital Sight DS-SM camera head. The total number of cells2-74