Sensitive detection of microRNAs using polyadenine-mediated fluorescent spherical nucleic acids and a microfluidic electrokinetic signal amplification chip

摘要

The identification of tumor-related microRNAs (miRNAs) exhibits excellent promise for the early diagnosis of cancer and other bioanalytical applications. Therefore, we developed a sensitive and efficient biosensor using polyadenine (polyA)-mediated fluorescent spherical nucleic acid (FSNA) for miRNA analysis based on strand displacement reactions on gold nanoparticle (AuNP) surfaces and electrokinetic signal amplification (ESA) on a microfluidic chip. In this FSNA, polyA-DNA biosensor was anchored on AuNP surfaces via intrinsic affinity between adenine and Au. The upright conformational polyA-DNA recognition block hybridized with 6-carboxyfluorescein-labeled reporter-DNA, resulting in fluorescence quenching of FSNA probes induced by AuNP-based resonance energy transfer. Reporter DNA was replaced in the presence of target miRNA, leading to the recovery of reporter-DNA fluorescence. Subsequently, reporter-DNAs were accumulated and detected in the front of with Nafion membrane in the microchannel by ESA. Our method showed high selectivity and sensitivity with a limit of detection of 1.3 pM. This method could also be used to detect miRNA-21 in human serum and urine samples, with recoveries of 104.0–113.3 and 104.9–108.0, respectively. Furthermore, we constructed a chip with three parallel channels for the simultaneous detection of multiple tumor-related miRNAs (miRNA-21, miRNA-141, and miRNA-375), which increased the detection efficiency. Our universal method can be applied to other DNA/RNA analyses by altering recognition sequences.