Enzyme-assisted electrochemical point-of-care test for miRNA detection towards liquid biopsy application

Liquid biopsy enables emerging biomarkers detection, such as circulating tumor DNA (ctDNA), microRNAs (miRNAs), and exosomes, leading to real-time monitoring of cancer progression and therapy effectiveness. Important circulating miRNAs, serving as biomarkers, have been correlated to breast cancer (BC) metastasis and therapy efficiency. In particular, among the BC, the Triple negative BC (TNBC) is an aggressive and heterogeneous subtype, accounting for 40% of BC related mortality. Rapid detection of these miRNAs through non-invasive liquid biopsy can significantly enhance diagnosis and prognosis, thereby improving survival rates. Considering these emerging biomarkers within the framework of personalized medicine, affordable and portable devices could enable quicker monitoring for cancer patients, including those in remote areas. In this context, we have developed an enzyme-assisted electrochemical point-of-care (POC) test. The proposed miRNA detection system is boosted by the use of duplex-specific nuclease (DSN) as the recognition element of miRNA. The DSN enzyme exhibits high selectivity to discriminate DNA-RNA heteroduplexes triggering isothermal target recycling and signal enhancement. This capability is crucial for overcoming the limitations often associated with detecting trace amounts of miRNA in biofluids. In our study, the specific probe for the miRNA target was labelled with a redox mediator, methylene blue, to enable the detection of enzymatic products via electrochemical measurements on screen-printed electrode. Optimization studies have been performed to obtain the enzymatic reaction in terms of probe and enzyme concentration. A calibration graph was obtained for various target concentrations ranging from 0.1 pM to100 nM, and a detection limit down to the fM level was achieved. Specificity studies were performed with random miRNA targets, and finally the device was applied in spiked commercial serum samples. These promising results suggest potential for developing novel class of POC tests towards the realization of an all-in-one sustainable platform for liquid biopsy applications.