Sensitive fluorescent detection of exosomal microRNA based on enzymes-assisted dual-signal amplification

The analysis of microRNAs (miRNAs) in exosomes offers significant information for a rapid and non-invasive diagnosis of cancer. However, the clinical utility of miRNAs as biomarkers is often hampered by their low abundance in exosomes. Herein, we develop a dual-signal amplification biosensor for the sensitive detection of exosomal miRNA-21 (miR-21). In the presence of a cognate target, it hybridizes with a biotin-modified capture probe (Cp) to form a DNA-RNA heteroduplex that serves as a substrate for duplex-specific nuclease (DSN). With the assistance of DSN, the Cps are enzymatically hydrolyzed and numerous DNA catalysts are released, leading to the first signal amplification. After magnetic isolation, the DNA catalyst remaining in the supernatant triggers a strand displacement reaction based on the nicking-assisted reactant recycling strategy, without depleting the reactants, to implement the second signal amplification. Using this dual-signal amplification concept, our biosensor achieves a limit of detection of miR-21 of 0.34 fM, with a linear range of 0.5-100 fM. The receiver operating characteristic curve generated during clinical sample analysis indicates that the exosomal miR-21 outperforms serum carcinoembryonic antigen in discriminating between patients with gastric cancer (GC) and patients with precancerous (PC) lesions (area under the curve: 0.89 versus 0.74, n = 40). Moreover, the proposed biosensor exhibits an 83.9% accuracy in classifying patients with GC or PC lesions and healthy donors using a confusion matrix. Furthermore, patients with GC with or without metastases are discriminated using the proposed biosensor. Our technology may expand the applications of DNA-based biosensor-enabled cancer diagnostic tools.