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Open AccessArticle

On the Electrochemical Detection of Alpha-Fetoprotein Using Aptamers: DNA Isothermal Amplification Strategies to Improve the Performance of Weak Aptamers

1
Departamento de Química Física y Analítica, Universidad de Oviedo, Av. Julián Clavería 8, 33006 Oviedo, Spain
2
Instituto de Investigación Sanitaria del Principado de Asturias, Avenida de Roma, 33011 Oviedo, Spain
*
Author to whom correspondence should be addressed.
Biosensors 2020, 10(5), 46; https://doi.org/10.3390/bios10050046
Received: 9 April 2020 / Revised: 27 April 2020 / Accepted: 28 April 2020 / Published: 30 April 2020
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 (Kd) values, in the subµM 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. View Full-Text
Keywords: aptamer; alpha-fetoprotein; dissociation constant; rolling circle amplification; terminal deoxynucleotidyl transferase aptamer; alpha-fetoprotein; dissociation constant; rolling circle amplification; terminal deoxynucleotidyl transferase
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Lorenzo-Gómez, R.; González-Robles, D.; Miranda-Castro, R.; de-los-Santos-Álvarez, N.; Lobo-Castañón, M.J. On the Electrochemical Detection of Alpha-Fetoprotein Using Aptamers: DNA Isothermal Amplification Strategies to Improve the Performance of Weak Aptamers. Biosensors 2020, 10, 46.

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