Sensitive mutant DNA biomarker detection based on magnetic nanoparticles and nicking endonuclease assisted fluorescence signal amplification

Based on the nicking endonuclease (NEase)-assisted target recycling and magnetic nanoparticle (MNPs) separation process via the streptavidin-biotin system, we developed a new, signal amplified and ultrasensitive fluorescent biosensor for the detection of mutant human p53 gene. The target mutant DNA hybridizes with the loop portion of a hairpin probe (HP) modified with biotin and a fluorescein isothiocyanate dye (FITC) at its 5' and 3' ends separately and forms a nicking site for NEase, which cleaves the HP and releases the target DNA. The released target DNA again hybridizes with the intact HP and initiates the DNA recycling process with the assistance of NEase, leading to the cleavage of a large number of HPs and detachment of the biotin labeled part with the FITC tagged signal portion. Only these cleaved fragments corresponding to target DNAs could remain in solution and function as a signaling flare, while the biotin labeled sequences including intact hairpin probes could be trapped and removed by the streptavidin coated MNPs. The developed method exhibits a detection limit as low as 198 fM and high discrimination efficiency toward a single-base mismatched sequence. Therefore, the novel NEase-amplified magnetic nanoparticle assay has great potential for sensitive and accurate detection of trace amounts of DNA in clinical diagnosis and biomedical research.