Design and synthesis of DNA origami biosensors for DNA and protein detection

DNA nanotechnology is a promising technique for DNA and protein sensors, owing to its affinity to biological molecules and to the possibility of precisely tuning geometry and functions to optimize the actuation and signal transduction. Specifically, one can design two- and three-dimensional nanometric shapes with DNA origami, where a long DNA strand is folded by hybridization of shorter DNA strands. While a large variety of biosensing techniques exists, the versatility and programmability of DNA nanotechnology could make it a valid competitor in this field, overcoming some of the previous limitations. This project aims to demonstrate a DNA strand sensor that exploits DNA origami as the bioreceptor and redox indicators as transducers. Starting from existing systems fabricated using single DNA strands (EDNA), a characterization process is established, introducing electrochemical techniques for quantitative analysis, measuring probe density via cyclic voltammetry and electron transfer rate via square wave voltammetry. This is obtained by adapting previous methods to the new design, at the same time creating a point of reference for future systems. Through similar means and measurement techniques, we establish a fabrication process that is studied from the choice of substrate to the validation of each preparation step. Together with the active elements on the gold electrode, a passivation layer must be deposited to remove background currents. Several of such self-assembled monolayers are prepared and characterized, evaluating their interaction with DNA. A fundamental result is then heavily labelling DNA origami with redox active molecules, here methylene blue, in order to increase the current shift per single binding event, increasing contrast and signal amplification, two key parameters in any sensor. When DNA origami are folded under heavy loading conditions, the redox indicators cause them to aggregate, rendering them unusable. Several possible causes are hypothesized and tested, until successful labelling is obtained. The device is finally demonstrated in a proof of concept design that can perform sensing of short DNA strands through the detachment of methylene blue labelled DNA origami from a gold electrode, leading to a decrease in current proportional to the number of binding events. This system is a first step in the desired direction and can further be expanded by use of different structures that will create a single molecule system, allowing for in vivo use, and that will be able to accommodate different analytes, such as larger proteins.