Highly Sensitive and Label-free Fluorescence Detection of DNA Based on Structure Switch of Hairpin Probe DNA to G-Quadruplex-based DNAzyme

A label-free fluorescent assay for highly sensitive detection of DNA was developed based on structure switch of hairpin DNA probe to a G-quadruplex. based DNAzyme triggered by target DNA. The hairpin DNA probe includes sequences that correspond to the base sequence of the G. quadruplex and to the the sequence complementary to the target DNA, respectively. The hairpin structure of the probe DNA is energetically favored over the G-quadruplex structure in the absence of target DNA, resulting in the protection of the G-quadruplex in an inactive hairpin configuration. The hybridization of target DNA to the loop domain opens the stem of the hairpin, resulting in the self. assembly of the uncaged G-quadruplex structure, which acts as a DNAzymatic label for the signal production and amplification in the presence of hemin. The peroxidase. like DNAzyme oxidizes non. fluoresecnt 10-acetyl. 3,7-Dihydroxypenoxa-zin (ADHP) to the florescent product by H2O2, giving rise to fluorescence emission. This allowed the utilization of the H2O2-ADHP fluorescent system for quantitative analysis of DNA. The experimental conditions were optimized as: pH 8. 0, 10 mmol/L K+, 0. 2 mu mol/L Hemin, 50 mu mol/L ADHP. The assay showed a linear relationship toward target DNA concentration in the range of 5.0 pmol/L-1.0 nmol/L, with a limit of detection of 3.0 pmol/L (S/N = 3). The assay exhibited good selectivity against single. base mismatched DNA.