基于阳离子型共轭聚合物与酶底物探针的磷酸酯酶检测新方法

Novel Method of Phosphatase Detection Based on Cationic Conjugated Polymer and Enzymatic Substrate Probe

建立了一种基于阳离子型共轭聚合物与酶底物探针的磷酸酯酶检测新方法.阳离子型共轭聚合物通过静电吸引与荧光素修饰的带负电荷的三磷酸腺苷结合,并发生荧光能量共振转移;当加入磷酸酯酶时,它可以催化底物三磷酸腺苷上的磷酸酯基团逐渐水解,得到不带电荷的腺苷,从而使阳离子型共轭聚合物得以释放,能量转移效率下降.实验结果表明,能量转移效率下降的程度与酶的浓度相关.该方法操作简单、响应速度快、灵敏度高,易于扩展至其它能催化底物电荷密度变化的酶的检测,而且还有望应用于对酶具有抑制作用的药物分子的筛选.

A novel method was developed for phosphatase detection based on cationic conjugated polymer （CCP） and enzymatic substrate. The CCP associated with the fluorescein-labeled anionic adenosine triphosphate（ATP） by electrostatic attractions and fluorescence resonance energy transfer（FRET） from the CCP to the fluorescein-labeled ATP occurred. When phosphatase was added, it catalyzed the hydrolysis of phosphate groups from its substrate, ATP, and the final product adenosine was neutral, which made the CCP detached from adenosine. Then the FRET efficiency decreased. The results show that the degree of FRET efficiency decrease is related to the concentration of phosphatase. This detection method has the advantages including easy operations, real-time response, and high sensitivity. It can also sensitively assay other enzymes that promote the changes of the charge density of substrates ; moreover, it provides a promising application in drug screening based on the inhibition of enzymatic activity.