Jandl Bernhard, Sedghiniya Sima, Carstens Annika, Astakhova Kira
Department of Chemistry, Technical University of Denmark, 206-207 Kemitorvet, 2800 Kgs Lyngby, Denmark.
Institute of Applied Synthetic Chemistry, TU Wien (Vienna University of Technology), Getreidemarkt 9, 1060 Wien, Austria.
ACS Omega. 2019 Aug 16;4(9):13889-13895. doi: 10.1021/acsomega.9b01586. eCollection 2019 Aug 27.
Cancer is a major health risk in the modern society that requires rapid, reliable, and inexpensive diagnostics. Because of the low abundance of cancer DNA in biofluids, current detection methods require DNA amplification. The amplification can be challenging; it provides only relative quantification and extends time and cost of an assay. Herein, we report a new oligonucleotide hybridization platform for amplification-free detection of human cancer DNA. Using a large PEG-capture probe allows rapid separation of the bound (mutant) versus unbound (wild type) DNA. Next, a supramolecular hydrogel forming peptide attached to a detection oligonucleotide probe serves as a signal amplification tool. Having screened multiple short peptides and fluorophores, we identified the system P1 + cyanine 3.5 that allows for sensitive quantitative detection of mutation L858R in oncogene. The peptide-fluorophore-based assay provides absolute target DNA quantification at the detection limit of 20 ng cancer DNA versus >500 ng for Cy3.5-labeled oligonucleotide in only 1 hour.
癌症是现代社会中的一项重大健康风险，需要快速、可靠且廉价的诊断方法。由于生物流体中癌症DNA的丰度较低，当前的检测方法需要进行DNA扩增。这种扩增可能具有挑战性；它仅提供相对定量，并且会延长检测的时间和成本。在此，我们报告了一种用于无扩增检测人类癌症DNA的新型寡核苷酸杂交平台。使用大型聚乙二醇捕获探针可快速分离结合的（突变型）与未结合的（野生型）DNA。接下来，连接到检测寡核苷酸探针上的形成超分子水凝胶的肽用作信号放大工具。在筛选了多种短肽和荧光团后，我们确定了系统P1 + 花青素3.5，该系统能够灵敏地定量检测癌基因中的L858R突变。基于肽 - 荧光团的检测方法在仅1小时内就能在20 ng癌症DNA的检测限下提供绝对靶标DNA定量，而Cy3.5标记的寡核苷酸的检测限则大于500 ng。
