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

Ramón Lorenzo-Gómez,Daniel González-Robles,Rebeca Miranda-Castro,María Jesús Lobo-Castañón,Noemí De-Los-Santos-Álvarez

doi:10.3390/bios10050046

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.

Highlights

Cancer management is a multifactorial decision-making task that relies on a combination of clinical evidences, biochemical parameters and tumor biomarker values
Since the expression of AFP is highly specific for the liver, it was found elevated in hepatocellular carcinoma (HCC) patients and proposed as a tumor biomarker
We have studied the affinity of two anti-AFP aptamers previously reported to have very different affinities

Summary

Introduction

Cancer management is a multifactorial decision-making task that relies on a combination of clinical evidences, biochemical parameters and tumor biomarker values. The cut-off values and recommended use are under continuous scrutiny and could be modified over the years. This is the case of alpha-fetoprotein (AFP), one of the two biomarkers suitable for large-scale screening in addition to the controversial prostate specific antigen [1]. Since the expression of AFP is highly specific for the liver, it was found elevated in hepatocellular carcinoma (HCC) patients and proposed as a tumor biomarker. AFP alone is not enough for HCC diagnosis, screening or surveillance [4]. That is, increased levels in benign liver pathologies and teratomas or low levels in small HCC [5], has prompted that US and European guidelines allow HCC surveillance with or without AFP control, in contrast

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