期刊
ANALYST
卷 140, 期 15, 页码 5082-5089出版社
ROYAL SOC CHEMISTRY
DOI: 10.1039/c5an01042j
关键词
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资金
- National Natural Science Foundation of China [21375017, 21205015, 21105012]
- National Science Foundation for Distinguished Young Scholars of Fujian Province [2013J06003]
- Key Project of Fujian Science and Technology [2013Y0045]
- Program for New Century Excellent Talents of Colleges and Universities in Fujian Province [JA13130, JA13088]
- Medical Elite Cultivation Program of Fujian, P.R.C [2014-ZQN-ZD-26]
- Program for Fujian University Outstanding Youth Scientific Research [JA11105, JA10295]
- Foundation of Fuzhou Science and Technology Bureau [2013-S-122-4]
- Education Department of Fujian Province [JA13149]
In this paper, we describe a novel label-free fluorescence method for microRNA-21 (miR-21) detection based on terbium (Tb3+) and duplex-specific nuclease (DSN) assisted target recycling. Capture probes (Cps), containing a target-binding part and a signal-output part, are immobilized on magnetic beads (MBs). In the presence of the target miR-21, it hybridizes with the target-binding part of a Cp to form a DNA-RNA heteroduplex. Due to the considerable cleavage preference for DNA in DNA-RNA hybrids, DSN hydrolyzes the target-binding part of the Cp while liberating the intact target miR-21 to hybridize with a new Cp and initiate the second cycle of hydrolysis. Eventually, through magnetic separation, only the signal-output part of the Cp could remain in solution and function as a signalling flare to increase the fluorescence intensity of Tb3+ dramatically. By employing the above strategy, this approach can gain an amplified fluorescent signal and detect as low as 8 fM miR-21 under the optimized conditions. Moreover, due to the high selectivity of DSN, the method shows little cross-hybridization among the closely related miRNA family members even at the single-base-mismatched level. Successful attempts were made in applying the approach to detect miR-21 in human cell lysate samples of breast cancer patients.
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