A Novel Surface Plasmon Resonance Biosensor for Enzyme-free and Highly Sensitive Detection of MicroRNA Based on Multi Component Nucleic Acid Enzyme (MNAzyme)-mediated Catalyzed Hairpin Assembly

MicroRNAs (miRNAs) are potentially useful biomarkers for early diagnosis of human diseases. Here, a simple surface plasmon resonance (SPR) biosensor has been developed for highly sensitive detection of miRNA by designing a new enzyme-free and isothermal amplification strategy, named multi component nucleic acid enzyme-mediated mismatched catalyzed hairpin assembly (MNAzyme-CHA). The partial MNAzymes co-recognized the target to form a stable active MNAzyme, which continued to digest multiple hairpin H0 substrates, concomitantly generating a lot of fragments. The H0 fragments could initiate the mismatched CHA cycles, resulting in the generation of massive hairpin H1-H2 complexes. As a result, the H1-H2 complexes and streptavidin were attached to the sensor surface, leading to a significantly amplified SPR signal readout. The established biosensor showed high sensitivity and selectivity with a wide dynamic range from 1 pM to 100 nM. It was also successfully applied to the determination of target miRNA spiked into human total RNA samples. Thus, this developed biosensing strategy presents a simple and stable platform toward sensitive and convenient miRNA detection, and has great potential in assays of many other nucleic acids analytes for biomedical research and early clinical diagnosis.