Gold nanoparticle-facilitated assembly via supernatant transfer as a colorimetric SELEX and sensing platform for...

Accurate and selective detection of cortisol, a key biomarker for stress and metabolic health, is essential for diagnostics and healthcare. Single-stranded DNA (ssDNA) aptamers hold great promise for small molecule detection, yet conventional systematic evolution of ligands by exponential enrichment (SELEX) methods are often hindered by invisible screening and restricted conjugation capabilities. Here, we report a rapid and straightforward approach for cortisol aptamer discovery and determination using gold nanoparticle-facilitated assembly via supernatant transfer (GNP-FAST) coupled with GNP-SELEX. These GNP-mediated visual processes enable efficient ssDNA library refinement in only 6 rounds of GNP-FAST, followed by rapid aptamer discovery within 12 rounds of GNP-SELEX, culminating in a highly selective colorimetric assay for cortisol. Comprehensive engineering and characterization, including molecular docking, circular dichroism spectroscopy, and isothermal titration calorimetry, validated the performance of the identified aptamers. Notably, a truncated 26-mer of the selected aptamer, CorApt5-T, exhibited high binding affinity (K_D^app≈210 nM) and specificity for cortisol through positive cooperativity on the GNP surface. Consequently, CorApt5-T enabled a simple and rapid GNP-based colorimetric assay for cortisol in saliva and serum, demonstrating more pronounced cortisol-driven structural changes and superior sensitivity when compared to previously reported cortisol aptamers. These findings demonstrate the potential of integrating GNP-based colorimetric assay with GNP-FAST and GNP-SELEX as a visible platform for developing biosensors targeting a broad range of small molecules.