期刊
BIOSENSORS-BASEL
卷 10, 期 5, 页码 -出版社
MDPI
DOI: 10.3390/bios10050046
关键词
aptamer; alpha-fetoprotein; dissociation constant; rolling circle amplification; terminal deoxynucleotidyl transferase
资金
- Spanish Government [FPU16/05670, RTI-2018-095756-B-I00]
- Principado de Asturias Government [IDI2018-000217]
- FEDER funds
Affinity characterization is essential to develop reliable aptamers for tumor biomarker detection. For alpha-fetoprotein (AFP), a biomarker of hepatocellular carcinoma (HCC), two DNA aptamers were described with very different affinity. In this work, we estimate the dissociation constant of both of them by means of a direct assay on magnetic beads modified with AFP and electrochemical detection on carbon screen-printed electrodes (SPCE). Unlike previous works, both aptamers showed similar dissociation constant (K-d) values, in the sub mu M range. In order to improve the performance of these aptamers, we proposed the isothermal amplification of the aptamers by both terminal deoxynucleotidyl transferase (TdT) and rolling circle amplification (RCA). Both DNA amplifications improved the sensitivity and also the apparent binding constants from 713 nM to 189 nM for the short aptamer and from 526 nM to 32 nM for the long aptamer. This improvement depends on the true affinity of the binding pair, which ultimately limits the analytical usefulness.
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