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 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.

Characterization of extracellular matrix (ECM) is becoming more and more important to decipher cancer progression. Constant remodeling results in ECM components degradation or unusual ECM accumulation that releases short fragments to the body fluids. These fragments might be potential cancer biomarkers but to detect them specific receptors are needed. In response to this demand, we present the first electrochemical aptamer-based competitive assay for the minor collagen XI, dysregulated in several carcinomas. It was performed on magnetic beads using enzymatic labeling. First, we selected the most appropriate tag for the aptamer (biotin or 6-carboxyfluorescein). The former yielded higher currents by chronoamperometry and it was used for the competitive assay. The collagen fragment, a 16mer peptide used as the target, was detected from 52 to 1000 nM with an RSD of about 5%. The LOD of the assay was estimated as 24 nM (44 ng/mL). The performance of the assay in serum diluted 1:2 was equivalent to the assay in PBS. The detection of α1 chain of human collagen XI was also possible in cell lysates and confirmed by aptacytofluorescence, which is promising as a new tool to validate this fragment as a cancer biomarker.

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.