Docking-aided rational tailoring of a fluorescence- and affinity-enhancing aptamer for a label-free ratiometric malachite green point-of-care aptasensor

Although nucleic acid aptasensors are increasingly applied in the detection of environmentally hazardous biomolecules, several formidable challenges remain with this technique because of their vulnerability, high cost and suboptimal sensitivity. Here, a docking-aided rational tailoring (DART) strategy was established at three levels and in two dimensions for the refinement of malachite green (MG) DNA aptamers. Guided by in silico molecular docking, coarse and fine tailoring were conducted at three levels each, to significantly enhance fluorescence activation intensity and binding affinity in two dimensions. Empowered by the results of the rational tailoring, a mechanistic view of the MG DNA aptamer-target interaction was thoroughly analyzed via four types of interactions. To meet the demand for point-of-care testing (POCT), a label-free and ratiometric fluorescent aptasensor was developed leveraging the tailored MG aptamer, based on the binding site competition-equilibrium effect via the introduction of a reference dye. This sensitive, specific, low-cost and rapid aptasensor subsequently demonstrated outstanding detection performance, achieving an ideal signal response range of 5 nmol·L−1 - 6 μmol·L−1 and a low limit of detection (LOD) of 1.49 nmol·L−1. The DART strategy and systematic exploration of the MG DNA luminescent aptamers herein will provide a valuable reference in the field of aptamer tailoring, biosensing and bioimaging. The proposed label-free ratiometric aptasensor also provides a highly generalizable strategy for hazardous biomolecular detection. [Display omitted] •A label-free and dual-signal output ratiometric aptasensor for MG detection was developed.•Dual functionalities of recognition and signal output were demonstrated by the tailored MG aptamer.•Fluorescence property and affinity were enhanced by docking-aided rational tailoring (DART) strategy.•Mechanical analysis of binding between MG and aptamer was explained via four types of interactions.•Sensitive and rapid POCT of MG was achieved, with a wide detection range and low LOD, 1.49 nmol·L−1.