Institute of Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, Hannover 30167, Germany.
Institute of Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, Hannover 30167, Germany; Department of Biotechnology and Food Engineering, Technion Israel Institute of Technology, Technion City, 32000 Haifa, Israel.
Talanta. 2017 Sep 1;172:199-205. doi: 10.1016/j.talanta.2017.05.037. Epub 2017 May 18.
Sensitive and specific detection and quantification of small molecules often remain challenging. We developed a novel magnetic bead-based aptamer-assisted real-time PCR (Apta-qPCR) assay to provide a versatile platform for quantification of small molecules. The assay has been realized for the detection of ATP as a model system. The assay relies on a combination of qPCR with the target-induced dissociation (TID) of ATP aptamer from an oligonucleotide, complementary to the ATP binding site of the aptamer. The complementary oligonucleotide was immobilized on deoxythymidine (dT)-modified magnetic beads (dT-beads) and hybridized with the aptamer. The presence of ATP resulted in dissociation of the aptamer from the dT-beads and the dissociated aptamer was quantified using qPCR. The Apta-qPCR assay was able to detect 17nM ATP with a broad dynamic range from 50nM to 5mM. The assay is label-free, and real-time PCR-based detection of aptamer facilitates high sensitivity. The presented method is highly versatile and can be applied to various aptamer-target pairs to allow detection of a broad range of target analytes.
灵敏和特异地检测和定量小分子化合物仍然具有挑战性。我们开发了一种新型的基于磁珠的适体辅助实时 PCR（Apta-qPCR）检测方法，为小分子化合物的定量提供了一个通用的平台。该方法已用于检测 ATP 作为模型系统。该检测方法依赖于 qPCR 与 ATP 适体与互补于适体的 ATP 结合位点的寡核苷酸的靶诱导解离（TID）的结合。互补寡核苷酸固定在脱氧胸苷（dT）修饰的磁珠（dT-珠）上，并与适体杂交。ATP 的存在导致适体从 dT-珠上解离，并用 qPCR 定量解离的适体。Apta-qPCR 检测方法能够检测到 17nM 的 ATP，动态范围从 50nM 到 5mM。该检测方法是无标记的，基于实时 PCR 的适体检测具有高灵敏度。所提出的方法具有高度的通用性，可应用于各种适体-靶对，以允许检测广泛的目标分析物。
