Exponential and efficient target-catalyst rolling circle amplification for label-free and ultrasensitive fluorescent detection of miR-21 and p53 gene

MicroRNAs (miRNAs) and p53 gene can serve as valuable biomarkers for the diagnosis of a variety of cancers. Nevertheless, although the development of the DNA nanostructure on the detection of cancer-related biomarkers was initially demonstrated several years ago, the challenges of developing simpler, cheaper, and multi-level detection DNA biosensors persist. Herein, based on the rolling circle amplification (RCA) coupled with the target-triggered skill, we have developed a well-designed detecting platform. In this study, the dumbbell-shaped probes (DPP) could be cyclized and initiated through targets, thus beginning the target-catalyst RCA (tc-RCA) reaction, therefore engendering numerous dumbbell probe amplicons (DPA). Thereafter the probe primers (PP) mutually complementary to the loop of DPA was introduced, leading to the branch strand displacement reaction (B-SDA). SYBR Green I can effectively bind to the amplified double-stranded structures as a fluorescent reporter. Altering the target-binding sequence of the DPP, this biosensor can also be applied to detect different biomarkers. As a consequence, target miR-21 and p53 gene can be detected down to 0.65 fM and 2.04 fM respectively with a wide dynamic range. Moreover, we have also achieved the qualitative detection of interesting targets in cell lysates as well as the complex biological substrates and compared the results with reverse transcription quantitative PCR (RT-qPCR), thereby indicating the potential application in clinical diagnosis and biomedical research.