Screening and engineering of broad-spectrum aptamers for construction of a dual-mode aptasensor for highly sensitive detection of macrolide antibiotics

Macrolide antibiotics (MAs) are widely used in the fields of medicine, animal husbandry and aquaculture. However, residues due to the overuse of MAs threaten human health and cause ecological and environmental pollution. Aptasensors offer a promising solution for the simultaneous detection of these residues. In this study, a broad-spectrum aptamer (F1-Apt1) with high affinity for MAs was successfully screened using Capture-SELEX technology. Through secondary structure analysis and molecular docking, a truncated 40-nt G-quadruplex-containing aptamer (F1-apt1-40) was obtained, with the dissociation constants (K d ) ranging from 2.62 to 10.72 nM for five MAs. Subsequently, using F1-apt1-40 as the recognition element, a dual-mode aptasensor for detection of MAs was developed. In this sensing configuration, the binding of the targets induces a conformational change in the G4 scaffold of the aptamer, which can be simultaneously reported by (i) colorimetric signal from suppression of the G4/hemin peroxidase-like activity, and (ii) SYBR Green I fluorescence enhancement. The two readouts can be conducted separately in a single reaction system without aptamer modification or complex instrumentation. The dual-mode aptasensor achieved the detection limits of 47.2 nM for colorimetric mode and 60.6 nM for fluorescence mode, respectively. Evaluation experiments using actual samples spiked with MAs achieved the recoveries ranging from 95.46 to 100.86% and the relative standard deviation ranging from 1.60% to 6.10%. Moreover, the entire detection process can be completed in 15 min at room temperature, which offers a low-cost, robust and field-deployable tool for multiplex macrolides surveillance.