College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
Department of Microbiology and Immunology, College of Medicine, Drexel University, Philadelphia, 19102, USA.
Anal Chim Acta. 2024 Nov 15;1329:343254. doi: 10.1016/j.aca.2024.343254. Epub 2024 Sep 17.
Aptasensors have been extensively utilized in target detection due to their advantages of high sensitivity and fast response. However, the reliability of the detection results of the single-mode aptasensor cannot be verified in time. Developing efficient detection methods with cross-validation capability is beneficial to achieving highly reliable detection. This study aims to design a colorimetric and fluorescent dual-mode aptasensor by skillfully engineering G-quadruplex assembly and rolling circle amplification for highly reliable IFN-γ detection.
The complexes of anti-IFN-γ aptamers and complement sequences (cDNA) were modified on the magnetic beads. In the presence of IFN-γ, the preferential combination of aptamers with IFN-γ resulted in the release of cDNAs. The cDNAs were collected by magnetic separation and then used as primers to trigger rolling circle amplification reaction to generate enriched G-quadruplexes. The G-quadruplex could be utilized to combine with hemin to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine for colormetric mode or to couple with the fluorogenic dye Thioflavin T for fluorescent mode. The developed dual-mode aptasensor displayed a linear range of 1-10000 pM with a detection limit of 0.406 pM for the colormetric mode and a range of 0.1-10000 pM with a detection limit of 0.037 pM for the fluorescent mode. Further, the designed aptasensor was applied to IFN-γ detection in serum samples and achieved satisfactory recoveries.
This innovative dual-mode detection strategy skillfully leverages the effective target-binding ability of aptamer, dual-function of the G-quadruplex and the signal amplifying ability of rolling circle amplification. This approach not only provides a reliable testing tool for the detection of IFN-γ, but also promotes the development of multimode sensing platforms.
由于具有高灵敏度和快速响应的优势，适体传感器已被广泛应用于目标检测。然而，单模式适体传感器的检测结果的可靠性无法及时得到验证。开发具有交叉验证能力的高效检测方法有利于实现高度可靠的检测。本研究旨在设计一种比色和荧光双模适体传感器，通过巧妙地设计 G-四链体组装和滚环扩增，用于高度可靠的 IFN-γ 检测。
反 IFN-γ 适体和互补序列（cDNA）的复合物修饰在磁性珠上。在 IFN-γ 存在的情况下，适体与 IFN-γ 的优先结合导致 cDNA 的释放。通过磁性分离收集 cDNA，然后将其用作引物触发滚环扩增反应以产生富集的 G-四链体。G-四链体可用于与血红素结合以催化 3,3',5,5'-四甲基联苯胺的氧化用于比色模式，或与荧光染料 Thioflavin T 结合用于荧光模式。所开发的双模适体传感器在比色模式下的线性范围为 1-10000 pM，检测限为 0.406 pM，在荧光模式下的线性范围为 0.1-10000 pM，检测限为 0.037 pM。此外，该设计的适体传感器用于血清样品中的 IFN-γ 检测，获得了令人满意的回收率。
这种创新的双模检测策略巧妙地利用了适体的有效靶结合能力、G-四链体的双重功能和滚环扩增的信号放大能力。这种方法不仅为 IFN-γ 的检测提供了可靠的测试工具，而且促进了多模式传感平台的发展。
