On-Chip Purification of Extracellular Vesicles for microRNA Biomarker Analysis

Extracellular vesicles (EVs) and their cargo are increasingly suggested as innovative biomarkers correlated to the diagnosis, progression and therapy of diseases like cancer. Several techniques have been developed for the specific separation of the different classes of EVs that give solutions enriched in vesicles, but still containing other unwanted components. New methods for a more efficient, reliable and automated isolation of EVs are therefore highly desirable. Here, microparticles with surfaces endowed with positive ions were exploited to separate vesicles from complex biological matrices. First, flat silicon oxide surfaces functionalized with different divalent cations were tested for their efficiency in terms of small EV capture. Small EVs pre-purified via serial ultracentrifugations were employed for these analyses. The two better-performing cations, i.e., Cu2+ and Ni2+, were then selected to functionalize magnetic microbeads to be inserted in microfluidic chips and evaluated for their efficiency in capturing EVs and for their release of biomarkers. The best protocol setup was explored for the capture of EVs from cell culture supernatants and for the analysis of a class of biomarkers, i.e., microRNAs, via RT-PCR. The promising results obtained with this on-chip protocol evidenced the potential automation, miaturization, ease-of-use and the effective speed of the method, allowing a step forward toward its integration in simple and fast biosensors capable of analyzing the desired biomarkers present in EVs, helping the spread of biomarker analysis in both clinical settings and in research.