Ultrasensitive fluorescence microRNA biosensor by coupling hybridization-initiated exonuclease I protection and tyramine signal amplification

Tyramine signal amplification (TSA) has made its mark in immunoassay due to its excellent signal amplification ability and short reaction time, but its application in nucleic acid detection is still very limited. Herein, an ultrasensitive microRNA (miRNA) biosensor by coupling hybridization-initiated exonuclease I protection and TSA strategy was established. Target miRNA is complementarily hybridized to the biotin-modified DNA probe to form a double strand, which protects the DNA probe from Exo I hydrolysis. Subsequently, horseradish peroxidase (HRP) is attached to the duplex via the biotin-streptavidin reaction and catalyzes the deposition of large amounts of biotin-tyramine in the presence of hydrogen peroxide (H2O2), followed by the conjugation of signal molecule streptavidin-phycoerythrin (SA-PE), which generates an intense fluorescence signal upon laser excitation. This method gave broad linearity in the range of 0.1 fM - 10 pM, yielding a detection limit as low as 74 aM. An increase in sensitivity of 4 orders of magnitude was observed compared to the miRNA detection without TSA amplification. This biosensor was successfully applied to the determination of miR-21 in breast cancer cells and human serum. By further design of specific DNA probes and coupling with the Luminex xMAP technology, it could be easily extended to multiplex miRNA assay, which possesses great application potential in clinical diagnosis.