基于氧化石墨烯的碱性磷酸酶非标记荧光检测新方法

Novel label-free fluorescent assay of alkaline phosphatase based on graphene oxide

利用氧化石墨烯(GO)的吸附、荧光猝灭性能结合外切酶反应,发展了一种非标记荧光检测碱性磷酸酶(ALP)活性的新方法。双链DNA的5'端磷酸根被ALP水解成羟基,抑制了Lambda核酸外切酶(λexo)的降解,再与SYBR Green I(SGⅠ)结合并加入GO,仍得到显著的荧光信号。无ALP时,5'磷酸化的双链DNA被λexo降解成单链DNA进而被GO吸附猝灭。结果表明,新方法对ALP的检测具有高灵敏度、高信噪比,线性范围为0.0001~0.01 U/mL,检测限低至0.00006 U/mL。

A novel label-free fluorescent assay of alkaline phosphatase(ALP)activity was developed by using the adsorption and fluorescence quenching properties of graphene oxide(GO)combined with exonuclease reaction.The 5'-end phosphate of double-stranded DNA(dsDNA)was hydrolyzed by ALP into hydroxyl group,which inhibited the degradation of Lambda exonuclease(λexo),and then combined with SYBR Green I(SGⅠ)and added GO,one still observed a significant fluorescence signal.In the absence of ALP,5'phosphorylated dsDNA was degraded into single-stranded DNA byλexo and quenched by GO adsorption.The results show that the new method has high sensitivity and high signal-to-noise ratio for ALP detection,with a linear range of 0.0001~0.01 U/mL and a detection limit of 0.00006 U/mL.