Improved method for Mica functionalization used in single molecule imaging of DNA with atomic force microscopy

Abstract

The modified procedure of 1-(3-aminopropyl)silatrane (APS) compound synthesis based on a new derivative (3‑aminopropyl)trimethoxysilane for the purpose of DNA immobilization for AFM single imaging is described. New reaction pathway based on kinetically driven reaction approach is described. Necessity of two‑step purification process is proved; ability of purified APS to provide four times smoother surfaces in comparison with a crude product is demonstrated. Various analytical methods such mass spectroscopy and ¹H NMR were used to show structure and enhanced purity of the APS product. APS mediates fixation of DNA molecules to mica substrates to be used for DNA imaging under Atomic Force Microscope. The use of an APS compound for simple and rapid silanization of mica surface is demonstrated. The advantages of APS‑based method are based mainly on low roughness of modified mica and homogeneous surface coverage by short sequence dsDNA (246 bp). The product obtained by the condensation reaction was purified in a two step process whose effectiveness was demonstrated not only by reduction of the silanized surface roughness, but also by mass spectroscopy (MS‑ESi), MALDI‑TOF method and proton magnetic resonance spectroscopy. Experiments demonstrate that 1‑(3‑aminopropyl)silatrane can be used to fix dsDNA molecules to a mica surface to be visualized by either the tapping mode or the force‑volume mode of AFM microscopy, as demonstrated by experiments. Moreover, necessity of advanced purification protocol is demonstrated by AFM based roughness measurements – pure vs crude APS product. The kinetics of APS‑layer aging, caused by silicon oxide growth on silanized layers, was studied by water contact angle measurements and is discussed.