Patterned and switchable surfaces for biomaterial applications

Chapter 3 – Comparison of the binding mode of plasmid DNA to allylamine plasma polymer and poly(ethyleneglycol) surfacescharacterised by UV-Vis quantification and restriction enzyme digestion with Ssp1and Sph1 (New England Biolabs) with subsequent agarose gel electrophoresis.3.2.4. DNA adsorptionThe adsorption of plasmid DNA to ALAPP surfaces in various buffers wasperformed in cuvettes by analysing the depletion of solution fluorescence. 0.01 Mphosphate buffer containing 100 ng/ml pEGFP-N1 was varied to have a pH in therange of 5-9, [NaCl] 0.01-3 M and ethanol content 0-50% (v/v). For thermodynamicstudies, 0.01 M phosphate buffer containing 50-500 ng/ml pEGFP-N1 at a pH of 7.4,[NaCl] 0.5 M was utilised. For kinetic studies, 0.01 M phosphate buffer containing50, 100 and 500 ng/ml pEGFP-N1 at a pH of 7.4, [NaCl] 0.5 M was utilised.Picogreen ® (excitation 502 nm, emission 523 nm) (Molecular Probes) was added tothe working solution at a 400x dilution to monitor [DNA] throughout. Standardcurves of Picogreen ® fluorescence versus [DNA] were formed for each solution used.Fluorescence measurements were performed using a LS 55 LuminescenceSpectrometer (Perkin Elmer Instruments). Samples were initially incubated at roomtemperature overnight to allow adsorption of DNA to the cuvette walls. ALAPPcoated silicon wafers of known area were then added to the cuvette and the solutionwas allowed to incubate again for 8 hr. A decrease in solution fluorescence after theaddition of ALAPP films was related to the adsorption of DNA to the film. The massof the solution was monitored throughout the experiment in order to take intoaccount the effects of solvent evaporation. DNA adsorption studies excludingthermodynamic studies were conducted with a single batch of Si-ALAPP sampleswith an identical ageing period (1 day) to exclude differences in surface chemistry3-104