Enhancement of the signal resolution in aptamer assay upon target binding by employing DNA probes and dual fluorophores

In this study, we aimed to significantly enhance the resolution of aptamer assays by using DNA probes and dual fluorophores. The resolution is defined as the smallest change in the target concentration that causes a reliable and distinguishable signal change. Therefore, resolution enhancement in the aptamer assay ensures improved analytical performance. We first examined four aptamer-probe combinations in the aptamer assay and investigated the resolution by observing a percentage signal response difference (% ΔAAD/SYBR). A large % ΔAAD/SYBR means a discernable signal change for a certain concentration difference, which indicates a high-resolution assay. Consequently, significant resolution enhancement (approximately 3–7 times) was achieved using two DNA probes. The improved analytical performance of the resolution-enhanced aptamer assay was further demonstrated by a wide working range of quantification (i.e., 10⁻²–10² mg/L, four orders of magnitude), a regression curve with improved fitness, and a lower limit of detection (LOD) response (2–6 times lower).