Development of a DNA aptamer selection method based on the heterogeneous sandwich form and its application in a colorimetric assay for influenza A virus detection

Abstract

To facilitate the exact diagnosis of multiple influenza virus types that are reported each year, the quantitative, sandwich-format enzyme-linked immunosorbent assay (ELISA) is widely used. In this method, enzyme-linked secondary antibodies or aptamers serve as detection reagents for signal amplification or colorimetric analysis. Aptamers have similar target affinities and specificities to antibodies, and they are relatively easy to label and can be applied to a variety of methods. For this reason, many quantitative analysis methods have recently been proposed that have a heterogeneous sandwich format and use an antibody with high specificity for a target and an aptamer that can be easily labeled for signal amplification. Aptamers obtained through the conventional systematic evolution of ligands by the exponential enrichment (SELEX) method have a limitation in the heterogeneous sandwich format assay because they often bind the antibody that acts as the capturing agent. In this paper, we introduce an effective method for selecting aptamers that increases the signal-to-noise ratio in a heterogenous sandwich-type immunosensor and confirms the efficiency of the selected aptamer candidates in the colorimetric assay. Using the proposed method, four aptamer candidates with K_d values ranging from 77.6 to 125.7 nM were obtained. Of them, INFA-apt4 showed the strongest binding affinity, with quantitative detection of nucleoprotein ranging from 1 ng mL⁻¹ to 300 ng mL⁻¹ and a minimum detection limit of 1.02 ng mL⁻¹, which was 10-fold more sensitive than the values offered by conventional antibody-based methods.