Microscopic diagnostics of DNA molecules on diamond

Dr. Bohuslav Rezek

Abstract

Devices based on deoxyribonucleic acid (DNA) molecules find growing use in genetics, medicine, and drug discovery due to their ability to detect sequences of nucleic acids via hybridization. For recognition of hybridization events, DNA molecules are immobilized on surfaces. Design of efficient DNA sensors requires detailed knowledge of DNA bonding, arrangement, and density on the surface as they strongly influence accessibility for ions, rate of hybridization, and base mismatch recognition. Atomic force microscopy (AFM) represents a powerful tool for resolving these parameters as it features high spatial resolution (< 1 nm) and can operate in liquids where DNA adopts natural conformations. [2] As a substrate for DNA, diamond has become attractive due to its favorable chemical and bio-compatible properties. Compared to other materials, such as silicon, SiO₂, or gold, it does not show noticeable degradation of the interface. [1]

In this work, properties of hybridized deoxyribonucleic acid (DNA) arrays on single-crystalline undoped and boron-doped diamonds were studied on a microscopic level by AFM and fluorescence microscopy in buffered electrolytic solutions. DNA was immobilized on diamonds which were functionalized (i) photochemically by amino-decene molecules (TFAAD) and (ii) electrochemically by nitrofenyl-diazonium molecules. DNA immobilization was studied on H-terminated as well as oxidized diamond surfaces. On H-terminated surfaces, AFM measurements detected compact DNA layers. By analyzing AFM height and phase contrast, the DNA layer thickness of 76 – 92 Ǻ was obtained. The layer thickness indicates tilted arrangement of DNA chains under the angle of 36°– 47°. Morphology of DNA layers exhibited long-range undulations of 20 Ǻ in height and a characteristic nano-roughness of 8 Ǻ. By introducing these data into a geometrical model, the DNA density of 6x10¹² cm^-2 was calculated. Ordering of the DNA layers was not significantly influenced by buffer salinity in the range of 1 – 300 mM. By applying force in contact-mode AFM, the DNA molecules were compressed and, after exceeding certain force threshold, removed from the diamond surface. The threshold forces were >45 nN on photo- and >76 nN on electro-chemically functionalized diamonds, indicating that DNA is attached more strongly than on gold substrates. DNA arrangement and bonding strength were similar on H-terminated and oxidized diamond surfaces. An exception was photochemically processed oxidized surface where only some non-covalently bonded DNA was found. The AFM data are correlated with fluorescence images and are discussed in terms of chemical, mechanical, and electronic properties of DNA, diamond, and linker molecules.

[1] W. Yang et al., Nature Mat. 1 (2002) 253.

[2] B. Rezek, D. Shin, T. Nakamura, C. E. Nebel, J. Am. Chem. Soc. 128 (2006) 3884.