Patterned and switchable surfaces for biomaterial applications

Andrew Hook – Patterned and switchable surfaces for biomaterial applicationsQuantitative studies were conducted in a custom-built electrochemical cell asdescribed previously [124]. ALAPP samples were clamped into the cell and 2 ml of200 ng/ml pEGFP-N1 solution in saline (0.1 M NaCl) 0.01 M phosphate buffer(PBS), pH = 7.4 was added over the sample. After 24 hr incubation at roomtemperature, a voltage was applied in a stepwise fashion (+1.5 V – -2V) overconsecutive 2 min intervals with the substrate as the working electrode, anAg/AgCl/saturated KCl reference electrode and a platinum auxiliary electrode. TheDNA concentration of the solution was measured using PicoGreen ® (ex 502 nm, em523 nm) (Molecular Probes) according to the manufacturer’s specifications beforeand after each voltage application. Solution removed for DNA concentration analysiswas replaced with fresh PBS. Fluorescence was detected using a Perkin ElmerInstruments Luminescence Spectrometer LS 55.2.2.9. Solid phase transfectionALAPP substrates used for transfection and cell growth experiments weresterilised by incubation in ethanol (70%) for 15 min after which they were washed insterile ultra pure water and allowed to air dry in a sterile laminar flow hood. Otherconsumables were sterilised by autoclaving for 15 min at 120 °C. Human embryonickidney cells (HEK 293) were cultured in Dulbecco’s modified eagle media (DMEM)containing 10% serum, penicillin and streptomycin and incubated at 37 °C, 5% CO 2and 60-70% humidity. Identical incubation conditions were used for transfectionexperiments. Transfections were conducted on ALAPP substrates with the plasmidpEGFP-N1. 0.5 l of a 240 ng/l pEGFP-N1 solution was spotted onto the substratesurface with a typical area of 1.7 mm 2 and allowed to air dry in a sterile laminarflow hood. After drying, the DNA spots were rehydrated with 1 l sterile ultra pure2-73