期刊
SENSORS AND ACTUATORS B-CHEMICAL
卷 260, 期 -, 页码 70-77出版社
ELSEVIER SCIENCE SA
DOI: 10.1016/j.snb.2017.12.203
关键词
Label-free; Fluorescence biosensor; Hyper-branched rolling circle amplification; Ligase; Polynucleotide kinase
资金
- Central Public Research Institutes Basic Funds for Research and Development (Agro-Environmental Protection Institute, Ministry of Agriculture) [22060302008029]
- Fundamental Research Funds for assessment of the quality and safety of agricultural products [22130109001003]
- National Natural Science Foundation of China [21728801]
- Natural Science Foundation of Tianjin [16JCYBJC19900]
- Chinese Ministry of Agriculture
Ligase and polynucleotide kinase (PNK) play important roles in DNA repair process. In this work, a simple, convenient and ultrasensitive fluorescence biosensor for label-free detection of ligase and PNK was proposed on the basis of target-triggered hyper-branched rolling circle amplification (HRCA). In the presence of PNK and ligase, the 5'-OH end of a linear Padlock DNA strand could be phosphorylated by PNK and then be converted by ligase into a circular template to trigger subsequent exponential HRCA reaction, producing amounts of double-stranded DNA (dsDNA) fragments, accompanied by the significant fluorescence increase of dsDNA-intercalating dye-SYBR Green I (SG1). In the absence of ligase or PNK, however, HRCA primer and uncyclized Padlock strands were completely digested by exonucleases, thus providing a low background for the sensing system. Therefore, by recording the fluorescence change of SG I, ligase and PNK activity could be facilely determined. The proposed biosensor exhibited excellent detection sensitivity for ligase and PNK activity with the detection limits of 3.4 x 10(-4) U/mL and 3.8 x 10(-4) U/mL, respectively. Such a sensing strategy could be easily extended to studies on inhibitors of these two enzymes, thus might offer a promising tool in clinical molecular diagnostics and cancer therapy. (C) 2018 Elsevier B.V. All rights reserved.
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