Amplification-Free and Label-Free Multiplexed Profiling of Extracellular Vesicle-Derived MicroRNA via Micropore Sensing Based on PNA-Functionalized Hydrogel Barcodes.

Extracellular vesicle (EV)-derived microRNA (miRNA) is a promising biomarker for various diseases, including cancer. However, the current EV-miRNA detection technologies, such as RT-qPCR and microarray, have depended on the complex amplification and labeling processes, which are not preferred for constructing an on-site diagnosis system. Herein, we present an EV-miRNA detection platform utilizing micropore sensing based on peptide nucleic acid (PNA)-functionalized hydrogel barcodes. Based on the low background signal and high affinity to the miRNA of the PNA probes, the breast cancer-related miRNAs (miR-21 and let-7a) can be detected with femtomolar sensitivities (481 and 551 fM) without any amplification and labeling steps by penetrating the target-captured barcodes into the pore and analyzing the electrical signal. By designing the geometrical codes of the particles, the multiplexed detection of miR-21 and let-7a can be implemented with high specificity and practically applicable recovery rates. Finally, we validate the clinical potential of the presented assay by differentiating the expression patterns of the plasma EV-derived miR-21 and let-7a between the breast cancer patients and healthy donors.