Willner Itamar, Patolsky Fernando, Weizmann Yossi, Willner Bilha
Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Talanta. 2002 Apr 1;56(5):847-56. doi: 10.1016/s0039-9140(01)00658-0.
Three different methods for the amplified detection of a single-base mismatch in DNA are described using microgravimetric quartz-crystal-microbalance as transduction means. All methods involve the primary incorporation of a biotinylated base complementary to the mutation site in the analyzed double-stranded primer/DNA assembly. The double-stranded assembly is formed between 25 complementary bases of the probe DNA assembled on the Au-quartz crystal and the target DNA. One method of amplification includes the association of avidin- and biotin-labeled liposomes to the sensing interface. The second method of amplified detection of the base mismatch includes the association of an Au-nanoparticle-avidin conjugate to the sensing interface, and the secondary Au-nanoparticle-catalyzed deposition of gold on the particles. The third amplification route includes the binding of the avidin-alkaline phosphatase biocatalytic conjugate to the double-stranded surface followed by the oxidative hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate to the insoluble product indigo derivative that precipitates on the transducer. Comparison of the three amplification routes reveals that the catalytic deposition of gold on the Au-nanoparticle/avidin conjugate is the most sensitive method, and the single-base mismatch in the analyzed DNA is detected with a sensitivity that corresponds to 3x10(-16) M.
本文描述了三种利用微重力石英晶体微天平作为传感手段，对DNA中的单碱基错配进行扩增检测的方法。所有方法都涉及在分析的双链引物/DNA组装体中，将与突变位点互补的生物素化碱基进行初步掺入。双链组装体是在组装于金石英晶体上的25个互补碱基的探针DNA与目标DNA之间形成的。一种扩增方法包括将抗生物素蛋白和生物素标记的脂质体与传感界面结合。第二种碱基错配的扩增检测方法包括将金纳米颗粒-抗生物素蛋白偶联物与传感界面结合，以及金纳米颗粒催化金在颗粒上的二次沉积。第三条扩增途径包括将抗生物素蛋白-碱性磷酸酶生物催化偶联物与双链表面结合，随后将5-溴-4-氯-3-吲哚磷酸氧化水解为不溶性产物靛蓝衍生物，该衍生物沉淀在传感器上。对这三种扩增途径的比较表明，金在金纳米颗粒/抗生物素蛋白偶联物上的催化沉积是最灵敏的方法，分析DNA中的单碱基错配的检测灵敏度可达3×10⁻¹⁶ M。
