Department of Chemistry, Tsinghua University, Beijing 100084, China.
The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology, the Graduate School at Shenzhen, Tsinghua University, Shenzhen, Guangdong 518055, China.
Sensors (Basel). 2017 Mar 21;17(3):650. doi: 10.3390/s17030650.
Breast cancer is one of the most frequently diagnosed cancers in females worldwide and lacks specific biomarkers for early detection. In a previous study, we obtained a selective RNA-cleaving Fluorogenic DNAzyme (RFD) probe against MDA-MB-231 cells, typical breast cancer cells, through the systematic evolution of ligands by exponential process (SELEX). To improve the performance of this probe for actual application, we carried out a series of optimization experiments on the pH value of a reaction buffer, the type and concentration of cofactor ions, and sequence minimization. The length of the active domain of the probe reduced to 25 nt from 40 nt after optimization, which was synthesized more easily and economically. The detection limit of the optimized assay system was 2000 MDA-MB-231 cells in 30 min, which is more sensitive than the previous one (almost 5000 cells). The DNAzyme probe was also capable of distinguishing MDA-MB-231 cell specifically from 3 normal cells and 10 other tumor cells. This probe with high sensitivity, selectivity, and economic efficiency enhances the feasibility for further clinical application in breast cancer diagnosis. Herein, we developed an optimization system to produce a general strategy to establish an easy-to-use DNAzyme-based assay for other targets.
乳腺癌是全球女性最常见的癌症之一，缺乏用于早期检测的特异性生物标志物。在之前的一项研究中，我们通过指数富集的配体系统进化（SELEX）获得了针对 MDA-MB-231 细胞（典型的乳腺癌细胞）的选择性 RNA 切割荧光 DNA 酶（RFD）探针。为了提高该探针在实际应用中的性能，我们对反应缓冲液的 pH 值、辅助因子离子的类型和浓度以及序列最小化进行了一系列优化实验。优化后，探针的活性域长度从 40 个核苷酸减少到 25 个核苷酸，这使得探针的合成更加容易和经济。优化后的检测系统的检测限为 30 分钟内 2000 个 MDA-MB-231 细胞，比之前的检测限（近 5000 个细胞）更灵敏。该 DNA 酶探针还能够特异性地区分 MDA-MB-231 细胞与 3 种正常细胞和 10 种其他肿瘤细胞。这种具有高灵敏度、选择性和经济性的探针提高了在乳腺癌诊断中进一步临床应用的可行性。在此，我们开发了一种优化系统，为建立易于使用的基于 DNA 酶的其他靶标分析方法提供了一种通用策略。
