Abstract
Affinity characterization is an essential but time-consuming task to develop reliable aptamers for tumor biomarker detection and is not always thoroughly addressed. For neutrophil gelatinase-associated lipocalin (NGAL), a potential biomarker of pancreatic cancer, two DNA aptamers were described with very different affinity. Likewise, another pair of DNA aptamers was developed with very different affinity for alpha-fetoprotein (AFP), a biomarker of hepatocellular carcinoma. In this work, we estimated the dissociation constant of these aptamers by means of a direct assay on magnetic beads modified with biomarker and electrochemical detection on screen-printed carbon electrodes. In order to improve the performance of these aptamers, we proposed the isothermal amplification of the aptamers for both biomarkers by rolling circle amplification (RCA). In the case of AFP aptamers, we also tried terminal deoxynucleotidyl transferase (TdT), a template-independent amplification. Both DNA amplifications improved the sensitivity and the apparent binding constants of the aptamers for the two cancer biomarkers. Nevertheless, this improvement depends on the true affinity of the binding pair, which ultimately limits their analytical usefulness.
Highlights
Aptamers are synthetic receptors of nucleic acid nature that can bind to almost any type of target
The strength of the aptamer-target affinity binding is typically characterized by the equilibrium dissociation constant, Kd, which determines the analytical usefulness of the aptamer
Among all isothermal DNA amplifications successfully applied to aptamer-based systems, rolling circle amplification (RCA) and terminal deoxynucleotidyl transferase based amplification (TdT) have turned out to be
Summary
Aptamers are synthetic receptors of nucleic acid nature that can bind to almost any type of target. The strength of the aptamer-target affinity binding is typically characterized by the equilibrium dissociation constant, Kd, which determines the analytical usefulness of the aptamer. The estimation of this parameter requires methodologies sensitive enough to quantify usual Kd values within pM-nM range. In this sense, aptamers can be directly coupled to DNA amplification techniques, transforming the aptamer-target recognition event into a nucleic acid amplification reaction [1]. The possibility of enhancing the performance of these receptors by implementation of isothermal DNA amplification, either RCA or TdT, was evaluated
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