Patterned and switchable surfaces for biomaterial applications

Andrew Hook – Patterned and switchable surfaces for biomaterial applicationsA2.2.4. AFM tip modificationOTR8 and NP-20 tips were modified with a 27 mer, amino-oligonucleotide(AOLG) (5’-amino TTT AGT TGA TTT GTG CTT CAG TGT GCT, Geneworks).Tips were initially cleaned in piranha solution (3:1, H 2 SO 4 :30% H 2 O 2 ) for 30 sbefore rinsing in water and drying under nitrogen. Tips were then incubated inmercaptobutyltrimethoxysilane (1% in toluene) for 10 mins under nitrogen at roomtemperature before washing with toluene, then water and drying under nitrogen.Complete reduction of the mercapto group was ensured by incubation of the tips withtris(2-carboxyethyl) phosphine hydrochloride (66.3 mM in PBS) for 5 min and thenwashing and drying under nitrogen. Tips were then added to 3-(maleimido)propionicacid N-hydroxysuccinimide ester (3.76 mM in 1:1 PBS:DMF) and incubated for 2 hrunder nitrogen at room temperature with occasional irritation before washing withwater and drying under nitrogen. Tips were then added to AOLG (1.1 pM in PBS)and incubated for 2 hr under nitrogen at room temperature before washing with waterand drying under nitrogen. Finally, tips were added to ethanolamine (0.55 M inwater) and incubated for 10 mins at room temperature under nitrogen to quench anynon-reacted NHS groups, before extensive washing with water and drying undernitrogen.A2.2.5. AFM force measurementsForce measurements were conducted between OTR8 and NP-20 AOLG modifiedand unmodified tips and the polymer array as well as control polymer surfaces. ANanoscope IIIa scanning probe microscope was used in contact mode, with the initialvertical deflection set to -1.5 V, scan rate of 1 Hz, a ramp size of 1 m. Force curveswere taken under fluid in Tris-EDTA (TE) buffer supplemented with 10, 5, 1 and 0F