Pavlov Valeri, Willner Itamar, Dishon Arnon, Kotler Moshe
Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Biosens Bioelectron. 2004 Nov 15;20(5):1011-21. doi: 10.1016/j.bios.2004.06.020.
Telomerase is considered as an important biomarker for cancer cells. Two different methods for the amplified electrochemical and microgravimetric quartz-crystal-microbalance detection of telomerase activity originating from HeLa cancer cells are described. One method involves the telomerization of a primer (1) linked to the electrode, in the presence of telomerase from HeLa cell extract and dNTP, followed by the hybridization of a biotin-labeled nucleic acid (2) that is complementary to the telomere repeat units. The subsequent binding of an avidin-alkaline phosphatase conjugate (3) that catalyzes the oxidative hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate (4) results in the precipitation of the insoluble product (5) on the electrode. The second method involves the telomerization of the primer (1) associated with the electrode, in the presence of the telomerase-containing HeLa cell extract and the dNTP nucleotide mixture that includes biotin-labeled dUTP. The telomerization leads to the labeling of the telomeres with biotin labels. The association of the avidin-alkaline phosphatase conjugate (3) to the biotin labels results in the biocatalyzed transformation of (4) to (5) and the formation of a precipitate on the electrode or the Au-quartz crystal. As numerous precipitate molecules are formed as a result of the formation of a single telomere, the methods represent routes for the amplified detection of telomerase activity. The formation of the precipitate on the respective transducers is probed by following the changes in the electrode resistance using chronopotentiometry, or by following the frequency changes of the piezoelectric quartz crystals. The amount of precipitate generated on the electrodes is controlled by the concentration of the HeLa cancer cells. The methods enable the detection of telomerase activity that is extracted from 1000 HeLa cancer cells.
端粒酶被认为是癌细胞的一种重要生物标志物。本文描述了两种用于放大检测源自HeLa癌细胞的端粒酶活性的电化学和微重力石英晶体微天平检测方法。一种方法是在存在来自HeLa细胞提取物的端粒酶和dNTP的情况下，使与电极相连的引物（1）进行端粒化，随后使与端粒重复单元互补的生物素标记核酸（2）杂交。随后结合的抗生物素蛋白 - 碱性磷酸酶缀合物（3）催化5 - 溴 - 4 - 氯 - 3 - 吲哚基磷酸酯（4）的氧化水解，导致不溶性产物（5）在电极上沉淀。第二种方法是在含有端粒酶的HeLa细胞提取物和包含生物素标记dUTP的dNTP核苷酸混合物存在的情况下，使与电极相关的引物（1）进行端粒化。端粒化导致端粒被生物素标记。抗生物素蛋白 - 碱性磷酸酶缀合物（3）与生物素标记的结合导致（4）生物催化转化为（5），并在电极或金 - 石英晶体上形成沉淀。由于单个端粒的形成会产生大量沉淀分子，这些方法代表了放大检测端粒酶活性的途径。通过计时电位法跟踪电极电阻的变化，或通过跟踪压电石英晶体的频率变化，来探测各个传感器上沉淀的形成。电极上产生的沉淀量由HeLa癌细胞的浓度控制。这些方法能够检测从1000个HeLa癌细胞中提取的端粒酶活性。
