基于G-四链体/血红素DNA酶的高催化活性以及λ核酸外切酶(λexo)的辅助放大功能, 以17个碱基的DNA片段作为模型分析物, 构建了一种快速、灵敏、特异性好的新型Luminol-H₂O₂-G-四链体/血红素DNA酶化学发光传感体系。考察了λexo用量、Luminol浓度、H₂O₂浓度、溶液p H值对体系化学发光强度的影响。在最佳实验条件下, 即:p H=9.0, 10 units λexo, 1.0×10^-3 mol/L Luminol, 3.0×10^-2 mol/L H₂O₂, 测得DNA在2.0×10^-12~8.0×10^-9 mol/L的浓度范围内与Luminol的相对化学发光强度之间呈现良好的线性关系, 检出限(3σ)为0.7×10^-13 mol/L。本方法可以区分不同错配的核酸序列,并可用于复杂生物样品的分析。

A new versatile chemiluminescence biosensing platform with G-Quadruplexes/Hemin DNAzyme and lambda exonuclease (lambda exo) assisted signal amplification was designed for sensitive detection of DNA. The system involved target DNA, phosphate-DNA, auxiliary DNA and hairpin DNA. The selective digestion of lambda exo to 5'-phosphorylated strand of phosphate-DNA-auxiliary DNA-target DNA duplex sandwich structure resulted in the release of target DNA and auxiliary DNA. The released target DNA hybridized with another phosphate-DNA-auxiliary DNA duplex to trigger the target DNA recycling, while the released auxiliary DNA hybridized with hairpin DNA to activate DNAzyme segments. The activated DNAzyme segments interacted with hemin to form stable G-Quadruplexes/Hemin DNAzymes that could catalyze H₂O₂-mediated oxidation luminol to produce chemiluminescence signals. In the presence of 10 nits lambda exo, 1.0 x 10^-3 M luminol, 3.0 x 10^-2 M H₂O₂ and pH 9.0 buffer solution, the relative chemiluminescence intensity of luminol was linearly related with the concentrations of target DNA in the range of 2.0 x 10^-12 M to 8.0 x 10^-9 M. This unique analysis strategy provided a detection limit down to 7.0 x 10^-14 M. The present method was successfully applied to the determination of target DNA in serum samples, which was also capable of discriminating mismatched DNA from perfectly matched target DNA with a high selectivity.