Nucleic Acid Sequence Detection by a Multi-Technique Approach

Abstract

The development of highly sensitive, specific, cheap and rapid laboratory diagnostics is an increasingly important challenge. Diagnosis of virus or various cancer diseases can be achieved through nucleic acid sequence detection, able to recognize specific short DNA or RNA sequences. DNA-based biosensors are excellent candidates, thanks also to their reusable property. A careful multi-technique characterization of DNA self-assembled monolayers (SAMs) represents a crucial step in order to optimize a sturdy sequence detection scheme based on helix-helix hybridization. In our work, we exploit molecular self-assembly on gold of single 22/28-bases DNA strands complementary to specific disease-related RNA or DNA sequences. The characterization is based on microscopic (Atomic Force Microscopy, AFM), spectroscopic (Spectroscopic Ellipsometry, SE, and X-rays Photoelectron Spectroscopy, XPS) and mass-sensitive (Quartz-Crystal Microbalance with Dissipation, QCM-D) methods, in order to investigate structural, optical and packing properties of DNA films. By AFM nanolithography, regularly defined micro-areas were depleted from molecules under a high tip load: the depth of the shaved area provides an accurate estimate of the film thickness. Specific sequence recognition has therefore been detected by monitoring changes in the film thickness upon hybridization. Through SE experiments it has been possible to monitor the optical thickness (influenced by both thickness and refractive index) and the fingerprints of the 260 nm DNA molecular absorption, allowing to evaluate the hybridization state of a DNA monolayer through a non-destructive and rapid method. Finally, XPS measurements provided useful information about surface coverage, allowing to distinguish between single strand and double strand DNA monolayers, while by QCM-D we could follow the dynamic process and, from the changes in mass/area ratio, evaluate the molecular density.