基于λ核酸外切酶辅助放大的化学发光传感器用于DNA的灵敏检测  

Sensitive and Selective DNA Detection Based on Lambda Exonuclease-Assisted Signal Amplification

作　　者：陈佳[1] 郭芸芸[1] 陈桥[2] 邱洪灯[1] 

机构地区：[1]中国科学院兰州化学物理研究所西北特色植物资源化学重点实验室/甘肃省天然药物重点实验室,兰州730000 [2]西南交通大学材料科学与工程学院,材料先进技术教育部重点实验室,成都610031

出　　处：《分析化学》2015年第10期1513-1519,共7页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(No.21405163);甘肃省科技计划(No.145RJYA271)资助~~

摘　　要：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 unitsλexo,1.0×10^-3mol/L Luminol,3.0×10^-2mol/L H2O2,测得DNA在2.0×10^-12~8.0×10^-9mol/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^-13mol/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（ λexo）-assisted signal amplification was designed for sensitive detection of DNA. The system involves target DNA,phosphate-DNA,auxiliary DNA and hairpin DNA. The selective digestion ofλexo to 5＇-phosphorylated strand of phosphate-DNA-auxiliary DNA-target DNA duplex sandwich structure results in the release of target DNA and auxiliary DNA. The released target DNA can hybridize with another phosphate-DNA-auxiliary DNA duplex to trigger the target DNA recycling,while the released auxiliary DNA hybridizes with hairpin DNA to activate DNAzyme segments. The activated DNAzyme segments interact with hemin to form stable G-quadruplexes / hemin DNAzymes that can catalyze H2O2-mediated oxidation of luminal to produce chemiluminescence signals. Under the optimal experiment conditions,eg,10 units λexo,1. 0 ×10^-3mol / L luminol,3. 0 × 10^-2mol / L H2O2 and buffer solution at p H 9. 0,the relative chemiluminescence intensity of luminol is linearly related with the concentrations of target DNA in the range of 2. 0 × 10^-12mmol /L to 8. 0 × 10^-9mol / L. The unique analysis strategy gives a detection limit down to 0. 7 × 10^-13mol / L（ 3σ）.The present method has been successfully applied to the determination of target DNA in serum samples,and is also capable of discriminating single-base mismatched DNA from fully complementary target DNA with a high selectivity.

关 键 词：λ核酸外切酶 G-四链体/血红素DNA酶 化学发光 DNA 

分 类 号：O657.3[理学—分析化学] TP212.2[理学—化学]