Surface plasmon field-enhanced fluorescence energy transfer is employed for sensitive optical readout of a reversible hairpin aptamer assay that is suitable for continuous monitoring of low-molecular-weight chemical analytes. A hairpin aptamer specific to adenosine and adenosine triphosphate with Alexa Fluor 647 fluorophore attached to its 5′ end was anchored via 3′ end thiol to a gold thin film. Molecular spacers were used to control the distance of the fluorophore from the surface in the aptamer “off” and “on” states. The specific binding of the target analyte changes the aptamer conformation, which alters the distance of the fluorophore from the gold surface and translates to variations in the detected fluorescence intensity. The plasmonically mediated fluorescence signal increases the measured signal-to-noise ratio and allows for real-time observation of the analyte binding. Theoretical as well as experimental study of the optical signal dependence on fluorophore orientation, design of spacers, and angular distribution of collected light is presented for rational design of the assay. The detected sensor signal increased by a factor as large as 23 upon switching the aptamer from the “off” to “on” state due to the hairpin opening associated with the specific capture of target analyte.

中文翻译：

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用于发夹适体测定读数的等离子场增强荧光能量转移

表面等离子体场增强的荧光能量转移用于可逆发夹适体测定的灵敏光学读数，该测定适用于连续监测低分子量化学分析物。腺苷和三磷酸腺苷特异的发夹适体，其5'末端带有Alexa Fluor 647荧光团，通过3'末端硫醇固定在金薄膜上。使用分子间隔子来控制在适体“关”和“开”状态下荧光团与表面的距离。目标分析物的特异性结合会改变适体构象，从而改变荧光团与金表面的距离，并转化为检测到的荧光强度的变化。等离子体介导的荧光信号增加了测得的信噪比，并允许实时观察分析物的结合。理论和实验研究的光信号依赖于荧光团的方向，间隔物的设计和收集的光的角度分布的理论研究的合理设计的分析。当将适体从“关闭”状态切换到“开启”状态时，由于与目标分析物的特定捕获相关的发夹开口的打开，检测到的传感器信号增加了多达23倍。