Amplification strategies in DNA based biosensors for miRNA detection

DNA biosensors attracted great attention thanks to the application perspectives in many fields such as environmental analysis, food safety and diagnostics. Nowadays, new diagnostic tools are required for the sensitive detection of biomarkers in human samples, towards the replacement or the integration of common lab-based detection methods. MicroRNAs are very interesting biomarkers for many reasons: their role and deregulation in pathological processes, their specific expression, and their stability in body fluids. The challenge in their detection is related to their short sequences, the low concentration and the high sequence homology between different miRNAs. Common lab-based techniques require complex preparation of the sample and they are often not sensitive enough. DNA biosensors represent an alternative to these techniques, providing new detection methods able to support the common analytical strategies or replace them. We worked towards the development and the application of new strategies to improve the sensitivity of DNA based biosensors for miRNA detection. We designed Hybridization Chain Reaction to detect specific miRNAs. After the characterization in solution, we worked towards the implementation on electrochemical sensing platform and in sensing based on Localized Surface Plasmon Resonance in order to evaluate the enhancement in sensitivity reachable with HCR, comparing the results. In parallel, we designed an alternative detection method employing a triple helix probe, involving a double hybridization chain reaction able to induce a greater amplification. In principle, this strategy would allow the combination of different transduction methods and the adaptation to multiplexed detection. We demonstrated the working principle of this method and performed preliminary experiments in electrochemical detection of microRNAs. In line with our aim, in addition, we explored alternative strategies to enhance the response of DNA based biosensors, for instance investigating the possibility to employ DNA templated Ag/Pt nanoclusters in sensing applications.