Sun Ting, Xia Ning, Liu Lin
College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang 455000, China.
Nanomaterials (Basel). 2016 Jan 18;6(1):20. doi: 10.3390/nano6010020.
We presented a strategy for fabricating graphene oxide (GO)-based fluorescent biosensors to monitor the change of phosphorylation state and detect phosphatase activity. By regulating the interaction between the negatively charged phosphate group and the positively charged amino residue, we found that GO showed different quenching efficiency toward the phosphorylated and dephosphorylated dye-labeled peptides. To demonstrate the application of our method, alkaline phosphatase (ALP) was tested as a model enzyme with phosphorylated fluorescein isothiocyanate (FITC)-labeled short peptide FITC-Gly-Gly-Gly-Tyr(PO₃)-Arg as the probe. When the negatively charged phosphate group in the Tyr residue was removed from the peptide substrate by enzymatic hydrolysis, the resulting FITC-Gly-Gly-Gly-Tyr-Arg was readily adsorbed onto the GO surface through electrostatic interaction. As a result, fluorescence quenching was observed. Furthermore, the method was applied for the screening of phosphatase inhibitors.
我们提出了一种制备基于氧化石墨烯（GO）的荧光生物传感器的策略，用于监测磷酸化状态的变化并检测磷酸酶活性。通过调节带负电荷的磷酸基团与带正电荷的氨基残基之间的相互作用，我们发现GO对磷酸化和去磷酸化的染料标记肽表现出不同的猝灭效率。为了证明我们方法的应用，以碱性磷酸酶（ALP）作为模型酶，用磷酸化的异硫氰酸荧光素（FITC）标记的短肽FITC-Gly-Gly-Gly-Tyr(PO₃)-Arg作为探针进行测试。当通过酶促水解从肽底物中去除Tyr残基中的带负电荷的磷酸基团时，所得的FITC-Gly-Gly-Gly-Tyr-Arg通过静电相互作用很容易吸附到GO表面。结果，观察到荧光猝灭。此外，该方法还用于磷酸酶抑制剂的筛选。
