Ratiometric measurement is a technique that can provide precise data and even quantitative detection. To carry out ratiometric measurements, it is necessary that the sensor molecule exhibits a large shift in its emission or excitation spectrum after reaction with the target molecule. Fluorescence resonance energy transfer (FRET) is one mechanism used to obtain a large spectral shift. In this study, our aim was to develop a ratiometric fluorescent sensor molecule for phosphodiesterase activity based on FRET. We designed and synthesized CPF4 with a coumarin donor, a fluorescein acceptor, and two phenyl linkers having the phosphodiester moiety interposed between them. In the emission spectrum of CPF4 in aqueous buffer excited at 370 nm, the emission of the coumarin donor was strongly quenched and the emission of the fluorescein acceptor was observed. This emission spectrum demonstrates that energy transfer from the coumarin donor to the fluorescein acceptor proceeds efficiently. Addition of a phosphodiesterase to an aqueous solution of CPF4 resulted in an increase in the donor fluorescence and a decrease in the acceptor fluorescence. CPF4 exhibited a large shift in its emission spectrum after the hydrolysis of the phosphodiester group by the enzyme. This large shift of the emission spectrum indicates that ratiometric measurements can be made by using CPF4. The method described in this paper for designing enzyme-cleavable sensor molecules based on FRET should be readily applicable to other hydrolytic enzymes.

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基于荧光共振能量转移的磷酸二酯酶活性酶切传感分子的设计与合成

比率测量是一种可以提供精确数据甚至定量检测的技术。为了进行比率测量，传感器分子必须在与目标分子反应后在其发射或激发光谱中表现出较大的偏移。荧光共振能量转移 (FRET) 是一种用于获得大光谱位移的机制。在这项研究中，我们的目标是开发一种基于 FRET 的用于磷酸二酯酶活性的比率荧光传感器分子。我们设计并合成了带有香豆素供体、荧光素受体和两个苯基连接体的 CPF4，它们之间插入了磷酸二酯部分。在 370 nm 激发的水性缓冲液中 CPF4 的发射光谱中，香豆素供体的发射被强烈猝灭，并观察到荧光素受体的发射。该发射光谱表明从香豆素供体到荧光素受体的能量转移有效进行。将磷酸二酯酶添加到 CPF4 的水溶液中导致供体荧光增加和受体荧光降低。磷酸二酯基团被酶水解后，CPF4 的发射光谱发生了很大的变化。发射光谱的这种大偏移表明可以使用 CPF4 进行比率测量。本文中描述的基于 FRET 设计酶可裂解传感器分子的方法应该很容易适用于其他水解酶。将磷酸二酯酶添加到 CPF4 的水溶液中导致供体荧光增加和受体荧光降低。磷酸二酯基团被酶水解后，CPF4 的发射光谱发生了很大的变化。发射光谱的这种大偏移表明可以使用 CPF4 进行比率测量。本文中描述的基于 FRET 设计酶可裂解传感器分子的方法应该很容易适用于其他水解酶。将磷酸二酯酶添加到 CPF4 的水溶液中导致供体荧光增加和受体荧光降低。磷酸二酯基团被酶水解后，CPF4 的发射光谱发生了很大的变化。发射光谱的这种大偏移表明可以使用 CPF4 进行比率测量。本文中描述的基于 FRET 设计酶可裂解传感器分子的方法应该很容易适用于其他水解酶。发射光谱的这种大偏移表明可以使用 CPF4 进行比率测量。本文中描述的基于 FRET 设计酶可裂解传感器分子的方法应该很容易适用于其他水解酶。发射光谱的这种大偏移表明可以使用 CPF4 进行比率测量。本文中描述的基于 FRET 设计酶可裂解传感器分子的方法应该很容易适用于其他水解酶。