A graphene oxide platform for the assay of DNA 3′-phosphatases and their inhibitors based on hairpin primer and polymerase elongation
- 1 January 2013
- journal article
- research article
- Published by Royal Society of Chemistry (RSC) in Journal of Materials Chemistry B
- Vol. 1 (3), 361-367
- https://doi.org/10.1039/c2tb00109h
Abstract
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−1 and 0.003 U mL−1, respectively. The proposed method is anticipated to find applications in the study of DNA damage repair mechanisms.Keywords
This publication has 28 references indexed in Scilit:
- Label-free optical bifunctional oligonucleotide probe for homogeneous amplification detection of disease markersBiosensors and Bioelectronics, 2011
- Sensitive and Rapid Screening of T4 Polynucleotide Kinase Activity and Inhibition Based on Coupled Exonuclease Reaction and Graphene Oxide PlatformAnalytical Chemistry, 2011
- Ultrasensitive and selective detection of mercury(II) in aqueous solution by polymerase assisted fluorescence amplificationBiosensors and Bioelectronics, 2011
- Development of a High-Throughput Screening Platform for DNA 3′-Phosphatases and Their Inhibitors Based on a Universal Molecular Beacon and Quantitative Real-time PCRChemistry – An Asian Journal, 2010
- Coassembly of Graphene Oxide and Nanowires for Large-Area Nanowire AlignmentJournal of the American Chemical Society, 2009
- Sensitive and Selective Label-Free DNA Detection by Conjugated Polymer-Based Microarrays and Intercalating DyeChemistry of Materials, 2008
- Identification of DNA 3‘-Phosphatase Active Site Residues and Their Differential Role in DNA Binding, Mg2+ Coordination, and CatalysisBiochemistry, 2004
- Production, Characterization, and Epitope Mapping of Monoclonal Antibodies Against Human Polydeoxyribonucleotide KinaseHybridoma, 2001
- Molecular Cloning of the Human Gene, PNKP, Encoding a Polynucleotide Kinase 3′-Phosphatase and Evidence for Its Role in Repair of DNA Strand Breaks Caused by Oxidative DamageOnline Journal of Public Health Informatics, 1999
- 3'-Phosphatase activity in T4 polynucleotide kinaseBiochemistry, 1977