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Improving the analytical performance of weak aptamers: DNA isothermal amplification approaches (registering DOI)

Cancer diagnosis based on serum biomarkers, so-called liquid biopsy, is the most desirable but also the most challenging approach to diagnose cancer. This method relies on the presence of specific biomarkers in the accessible body fluids of cancer patients. However, many of these biomarkers are glycoproteins found in low concentration levels in a high protein content medium, the serum, thus hindering their detection. For this reason, biomarker detection requires strategies to boost sensitivity. Nucleic acid-based receptors such as aptamers are especially appropriate for this task because they can be easily manipulated with molecular biology tools such as polymerases and ligases, so they can be integrated in analytical schemes forbidden to other receptors like antibodies.

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 (HCC). In this work, we estimate 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 (SPCE). 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.

Keywords: aptamer characterization; neutrophil gelatinase-associated lipocalin; alpha-fetoprotein; rolling circle amplification; terminal deoxynucleotidyl transferase