Department of Biomedical Engineering, University of California, Davis, California 95616, USA.
Anal Chem. 2010 Mar 1;82(5):1851-7. doi: 10.1021/ac9025237.
Traditional antibody-based affinity sensing strategies employ multiple reagents and washing steps and are unsuitable for real-time detection of analyte binding. Aptamers, on the other hand, may be designed to monitor binding events directly, in real-time, without the need for secondary labels. The goal of the present study was to design an aptamer beacon for fluorescence resonance energy transfer (FRET)-based detection of interferon-gamma (IFN-gamma)--an important inflammatory cytokine. Variants of DNA aptamer modified with biotin moieties and spacers were immobilized on avidin-coated surfaces and characterized by surface plasmon resonance (SPR). The SPR studies showed that immobilization of aptamer via the 3' end resulted in the best binding IFN-gamma (K(d) = 3.44 nM). This optimal aptamer variant was then used to construct a beacon by hybridizing fluorophore-labeled aptamer with an antisense oligonucleotide strand carrying a quencher. SPR studies revealed that IFN-gamma binding with an aptamer beacon occurred within 15 min of analyte introduction--suggesting dynamic replacement of the quencher-complementary strand by IFN-gamma molecules. To further highlight biosensing applications, aptamer beacon molecules were immobilized inside microfluidic channels and challenged with varying concentration of analyte. Fluorescence microscopy revealed low fluorescence in the absence of analyte and high fluorescence after introduction of IFN-gamma. Importantly, unlike traditional antibody-based immunoassays, the signal was observed directly upon binding of analyte without the need for multiple washing steps. The surface immobilized aptamer beacon had a linear range from 5 to 100 nM and a lower limit of detection of 5 nM IFN-gamma. In conclusion, we designed a FRET-based aptamer beacon for monitoring of an inflammatory cytokine-IFN-gamma. In the future, this biosensing strategy will be employed to monitor dynamics of cytokine production by the immune cells.
传统的基于抗体的亲和传感策略需要使用多种试剂和洗涤步骤，不适合实时检测分析物结合。另一方面，适体可以设计为直接实时监测结合事件，而无需使用二级标记。本研究的目的是设计一种适体荧光共振能量转移（FRET）的生物传感器用于检测干扰素-γ（IFN-γ）-一种重要的炎症细胞因子。经过修饰带有生物素部分和间隔物的 DNA 适体变体被固定在亲和素涂层表面，并通过表面等离子体共振（SPR）进行了表征。SPR 研究表明，通过 3' 端固定适体导致与 IFN-γ的最佳结合（K(d)=3.44 nM）。然后，使用这种最佳适体变体通过将荧光标记的适体与带有淬灭剂的反义寡核苷酸链杂交来构建生物传感器。SPR 研究表明，在引入分析物 15 分钟内，与适体生物传感器发生 IFN-γ结合，表明 IFN-γ分子动态取代了淬灭剂互补链。为了进一步强调生物传感应用，将适体生物传感器分子固定在微流控通道内，并与不同浓度的分析物进行了挑战。荧光显微镜显示在没有分析物的情况下荧光较低，而在引入 IFN-γ后荧光较高。重要的是，与传统的基于抗体的免疫分析不同，该信号是在结合分析物时直接观察到的，而无需进行多次洗涤步骤。表面固定的适体生物传感器的线性范围为 5 至 100 nM，检测下限为 5 nM IFN-γ。总之，我们设计了一种基于 FRET 的适体生物传感器用于监测炎症细胞因子 IFN-γ。在未来，这种生物传感策略将用于监测免疫细胞产生细胞因子的动态。
