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

Abstract

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.

Improving the Analytical Performance of Weak Aptamers: DNA Isothermal Amplification Approaches

“…In the case of AFP, the affinity of two different aptamers, AFP-S and AFP-L, was studied. AFP-S was adapted to the RCA scheme, as previously mentioned, and this amplification produced a 6fold improvement in the apparent Kd (188 ± 33 nM) in comparison with the non-RCA amplified assay (713 ± 63 nM) [9]. These affinity values were close to those previously reported (500 nM) [7], but the improvement attained with the amplification was much modest than the one achieved with the anti-NGAL aptamer.…”

“…On the other hand, AFP-L was amplified by TdT under optimized conditions. In this case, the TdT-amplified assay led to a 16-fold enhancement of the apparent Kd (32 ± 11 nM) compared to the non-amplified assay (526 ± 101 nM) [9]. These apparent Kd values were above the previously reported one (2.37 nM) [6], indicating that the "real" affinity of this aptamer is weaker than expected.…”

“…First, we studied the biotin-dATP/dATP ratio to obtain the longest tail with a proper number of labels in solution. The elongated aptamers were analyzed by gel electrophoresis, and it was revealed that higher concentrations of labeled nucleotide led to shorter products [9].…”

“…To strike a balance between enhancing the reaction rate (more unlabeled nucleotides) and the electrochemical signal (more labeled nucleotides), a concentration of 25 µ M of biotin-dATP was chosen as optimum. Since the rate of elongation is nucleotide dependent, we also studied the effect of using only dATP or an equimolar mixture of the 4 deoxyribonucleotides (dNTPs), and found that the mixture produced higher specific currents [9].…”

A Paper-Based Photoelectrochemical Sensing Platform Based on In Situ Grown ZnO/ZnIn2S4 Heterojunctions onto Paper Fibers for Sensitively Detecting AFP

“…Alpha-fetoprotein (AFP), as one of the most basilic tumor biomarkers, with a molecular weight about 70,000 Da, is normally used for routine cancer screening in clinical laboratories because it is universally associated with many cancer incidences, including gastric, breast, colorectal, and liver cancers [ 26 , 27 , 28 , 29 ]. Accordingly, it is valuable to explore sensitive, accurate, and effective bioassays strategies in biological samples combined with various techniques for AFP determination.…”