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

ABSTRACT

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 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 H2 O2 , giving rise to fluorescence emission. This allowed the utilization of the H2 O2-ADHP fluorescent system for quantitative analysis of DNA. The experimental conditions were optimized aspH 8. 0, 10 mmol/L K+, 0. 2 μmol/L Hemin, 50 μmol/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.