I have been successful in improving and developing a homemade device based on a three-electrodes electrochemical redout setup thanks to it, we are able to perform measurements of DNA-hybridization, in real-time, from probe ssDNA-SAMs coupled to gold coated sensor surfaces. The measurements were carried out, in pure saline buffer solution, on a large range of concentrations of complementary-DNA strands (from 1 pM to 100 nM), monitoring the differential capacitance at the Working Electrode versus the incubation time. The studies on kinetics, modeled using the Langmuir adsorption model, not only give us important information on the kinetics itself but they allow us to detect eventual mismatches along the DNA-sequence target proving to be sensitive to the position of the mismatch with respect to the surface of the device or to define, in human extract and plasma, the unknown concentration of a specific miRNA-target connected to the heart failure taking into account the hindrances carried by the Argonaute proteins in which the miRNA are inglobed. This goal was achieved by performing a calibration curve on experiments of DNA/DNA hybridization performed in a simple saline buffer. The results were then confirmed using a real time qPCR by our partners in MD D. Cesselli's and MD A.P. Beltrami's group at University of Udine. Another strand of my PhD project concerns the detection of more complex components such as proteins or single-domain antibodies (e.g. VHH fragments)–DNA conjugates, with the final purpose of the detection of circulating tumor cells (CTCs) not only in pure saline buffer but also in human serum. In particular, we have focused on the detection of the protein HER2 whose overexpression is connected to certain aggressive types of breast cancer. In addition, the systematic characterization of the device caught our attention, and it was developed by performing measurements of Self Assembled Monolayer (SAM) detection, carried out in different physiological buffers (KCl, NaCl, MgCl2, PBS, etc.), in different probe-density conditions and applying different potential in order to have a more comprehensive understanding of the phenomena occurring at the electrode/electrolyte interface. Studies that have led to the implementation of a theoretical model, able to provide an acceptable physical explanation of the biorecognition events of interest.
