Kennedy, J. (2019). PREPARATION AND VISUALIZATION OF INDIVIDUAL ACTIN FILAMENTS USING ATOMIC FORCE MICROSCOPY FOR THE STUDY OF ACTIN/DNA HYDROGEL COMPLEXES. Unc Charlotte Electronic Theses And Dissertations.
A protocol of sample preparation for the visualization of single actin filaments at the nanoscale using atomic force microscopy for use in studies of engineered actin/DNA conjugated hydrogel complexes was developed. Hydrogels are cross-linked networks of hydrophilic monomers synthesized into polymers which behave as solids at the macroscale but exhibit fluid-like properties at the microscale, containing interstitial spaces which swell with water, allowing water content to multiply to thousands of times their weight, resulting in viscoelastic mechanical properties. F-actin and DNA have both exhibited potential for use in biocompatible engineered hydrogel applications. A procedure for actin sample preparation was developed which utilized aminopropyltriethoxysilane (APTES) to covalently attach a charged amine group (N, N-diisopropylethylamine) to mica substrates through vapor deposition in a desiccator that had been purged with nitrogen before deposition of F-actin samples and imaging. This method reliably produced images of individual actin filaments suitable for studies of engineered DNA/actin hydrogels. In addition, initial work was begun on the formation of two different potential DNA/actin conjugate complexes using complementary DNA oligomers combined with crosslinking molecules (SMPB) and actin. Initial results showed differences in average filament diameters and average persistence lengths in conjugate samples compared to actin samples.