期刊
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
卷 409, 期 28, 页码 6677-6688出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-017-0623-0
关键词
Electrochemical biosensor; Terminal deoxynucleotidyl transferase; BamHI; Copper nanoclusters; Graphene oxide
资金
- National Natural Science Foundation of China [41576098, 81273130, 21605089]
- Open Subject of State Key Laboratory of Chemo/Biosensing and Chemometrics [2016001]
- Project of Academic Research Foundation of Ningbo University [XYL17002]
- Ningbo Municipal Natural Science Foundation [2016A610052]
- Science and Technology Department of Zhejiang Province of China [2016C33176, 2016R405068]
- K.C. Wong Magna Fund in Ningbo University
Electrochemical methods allow fast and inexpensive analysis of enzymatic activity. Here, a simple and yet efficient signal-on electrochemical assay for sensitive, label-free detection of DNA-related enzyme activity was established on the basis of terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. TdT, which is a template-independent DNA polymerase, can catalyze the sequential addition of deoxythymidine triphosphate (dTTP) at the 3'-OH terminus of single-stranded DNA (ssDNA); then, the TdT-yield T-rich DNA nanowires can be employed as the synthetic template of copper nanoclusters (CuNCs). Grown DNA nanowires-templated CuNCs (noted as DNA-CuNCs) were attached onto graphene oxide (GO) surface and exhibited unique electrocatalytic activity to H2O2 reduction. Under optimal conditions, the proposed biosensor was utilized for quantitatively monitoring TdT activity, with the observed LOD of 0.1 U/mL. It also displayed high selectivity to TdT with excellent stability, and offered a facile, convenient electrochemical method for TdT-relevant inhibitors screening. Moreover, the proposed sensor was successfully used for BamHI activity detection, in which a new 3'-OH terminal was exposed by the digestion of a phosphate group. Ultimately, it has good prospects in DNA-related enzyme-based biochemical studies, disease diagnosis, and drug discovery.
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