We have developed a label-free, simple and highly sensitive hairpin fluorescent biosensor for the assay of DNA 3'-phosphatases and their inhibitors utilizing a graphene oxide (GO) platform. In this assay, we designed a hairpin primer (HP) with a 3'-phosphoryl end that served as the substrate for DNA 3'-phosphatases. Once the phosphorylated HP was hydrolyzed by DNA 3'-phosphatases, the resulting HP with a 3'-hydroxyl end was immediately elongated to form a long double-strand product by Klenow fragment polymerase (KF polymerase). With SYBR green I (SG) selective staining of the double-helix DNA, a very high fluorescence enhancement was achieved. Furthermore, GO was introduced to quench the fluorescence of the HP without polymerase elongation, thereby further increasing the signal-to-background ratio. The proposed method is simple and convenient, yet still exhibits high sensitivity and selectivity. This method has been successfully applied to detecting the activity of two typical 3'-phosphatases, T4 polynucleotide kinase phosphatase (PNKP) and shrimp alkaline phosphatase (SAP). The effect of their inhibitors has also been investigated. The results revealed that the method allowed a sensitive quantitative assay of T4 PNKP and SAP, with detection limits of 0.07 U mL and 0.003 U mL, respectively. The proposed method is anticipated to find applications in the study of DNA damage repair mechanisms.
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