Enzyme-Assisted Electrochemical Point-of-Care Test for miRNA Detection in Liquid Biopsy

In the personalized medicine era, affordable and portable devices for quicker cancer monitoring, even in remote areas, are crucial. To address this need, we have developed an enzyme-assisted electrochemical point-of-care (POC) test for application toward liquid biopsy. In particular, miR-200a-5p has been taken into account as the model target due to its correlation for triple negative breast cancer (TNBC) prognosis. The proposed platform has been conceived as signal-ON, and the detection architecture is based on the presence of a duplex-specific nuclease (DSN) that is selective for DNA-RNA heteroduplexes. When the miRNA is recognized by an ad-hoc designed DNA probe, the DSN enzyme enables for the isothermal target recycling and signal enhancement, which is essential for detecting the miRNA trace in biofluids. Introducing a methylene blue (MB) modification on the DNA probe, a single miRNA strand is capable of triggering multiple DSN cleavage circles, increasing the free MB and thus the electrochemical signal recorded. All the optimization studies have been carried out using a screen-printed strip, resulting in a dynamic range comprised between 0.1 pM and 100 nM and a detection limit down to the fM level. A satisfactory selectivity was highlighted by interrogating the system toward random miRNA target mixtures, and the platform was also tested in spiked commercial serum samples.