Patterned and switchable surfaces for biomaterial applications

Andrew Hook – Patterned and switchable surfaces for biomaterial applicationsinteractions between the ALAPP and the adsorbed DNA must be overcome by asuitable potential before the DNA can be released into solution. A plateau in therelease of DNA was observed for increased negative voltages above -500 mV(Figure 2.7), thus, for subsequent experiments -750 mV was selected as a suitablevoltage to stimulate the desorption of adsorbed DNA in order to minimise the voltagerequired to desorb the DNA whilst ensuring DNA desorption was maximised. Intotal, 85% of the adsorbed plasmid DNA was released by application of the negativevoltage, with a final DNAof 0.18 mg/m 2 . Once conditions for the controlledadsorption and desorption of surface-bound plasmid DNA were established,experiments were designed to test for its bioavailability. This was done by carryingout transfection assays.2.3.3. Transfection experimentsThe ultimate test of the concept investigated here was the ability of the surface toallow solid phase transfection with reasonable efficiencies. HEK 293 cells weregrown on ALAPP films after adsorption of plasmid DNA (pEGFP-N1) in thepresence of a transfection agent (Effectene). In order to mimic the transfected cellmicroarray plasmid DNA was manually spotted onto the ALAPP surface, as opposedto incubating the ALAPP in plasmid solution for 24 hr. Transfected cells showedgreen fluorescence. The total cell population was visualised using Hoechst 33342 bythe blue fluorescence of the minor groove binder, which is incorporated into thenucleus of all living cells, non-transfected and transfected. Transfection efficiencieswere determined by dividing the number of transfected cells by the total number ofcells present. In order to study the effect of applying a negative voltage and electrostimulatingthe desorption of DNA on transfection efficiency, first 0, -250, -500, -2-89