DNA aptamer-based bioanalysis of IgE by fluorescence anisotropy
DNA aptamer-based bioanalysis of IgE by fluorescence anisotropy
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DOI：
10.1021/ac0483926
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发表时间：
2005-04-01
影响因子：
7.4
通讯作者：
Wilson, GS
中科院分区：
文献类型：
--
作者：
Gokulrangan, G;Unruh, JR;Wilson, GS
报道了一种快速、均一的基于适配子的生物分析方法，用于荧光偏振(FP)法灵敏地检测免疫球蛋白E(IgE)。根据标记配体的各向异性差异，用5‘端标记的D17.4DNA适配子检测IgE。用荧光素和德克萨斯红两种不同的荧光试剂分析低纳摩尔范围的IgE，具有很高的特异性。在较短的平衡时间内，观察到了可测量的各向异性变化。对结合数据的分析揭示了溶液中可能的协同结合过程。荧光团的性质明显影响分析的灵敏度，而不是用于染料偶联的系绳长度。局部荧光团的运动被认为对FP探头的灵敏度有显著影响。德克萨斯红被认为对这种方法相对更敏感，并具有明显有利的染料-DNA相互作用，检测极限达到350 PM。在这项工作中，已经观察到FP响应的显著的温度依赖性。结合缓冲液的离子组成也影响测定的灵敏度。NE结果证实了类似的均相分析在基于适体的生物分析中的前景和潜力。
A rapid, homogeneous aptamer-based bioanalysis is reported for the sensitive detection of immunoglobulin E (IgE) using fluorescence polarization (FP). 5 '-End-labeled D17.4 DNA aptamer was used for IgE detection based on the anisotropy differences of the labeled ligand. Two different fluorophores, fluorescein and Texas Red, were used to analyze IgE in the low-nanomolar range with high specificity. Measurable anisotropy changes were observed with a short equilibration time. Analysis of the binding data reveals a possible cooperative binding process in solution. The nature of the fluorophore clearly influences the sensitivity of the analysis more than the tether length used for the dye conjugation. The local fluorophore motion is seen to influence the sensitivity of the FP probe significantly. Texas Red is seen to be relatively more sensitive for this approach and has apparently favorable dye-DNA interactions, and a limit of detection of 350 pM was obtained. Significant temperature dependence of the FP responses has been observed in this work. Ionic composition of the binding buffer also influences the assay sensitivity. ne results confirm the promise and potential of similar homogeneous assays for aptamer-based bioanalysis.