Screening and application of a truncated aptamer for high-sensitive fluorescent detection of metronidazole

Abstract

Aptamer-based biosensors have been widely constructed and applied to detect diverse targets. Metronidazole is a widely used broad-spectrum antibacterial drug, whose residue has multiple risks to human health. Herein, metronidazole-specific aptamers were selected from a random ssDNA library with the full length of 79 nucleotides (nt) based on DNA library-immobilized magnetic beads SELEX technology. After ten rounds of selection, four aptamers with highly similar secondary structures were selected, among which AP32, with the lowest dissociation constants, was chosen as the optimal aptamer for further optimization. Then a semi-rational post-SELEX truncation was carried out based on the secondary structure analysis and molecular docking, as well as affinity assessment. Redundant nucleotides in AP32 were stepwise removed without the decrease of affinity. Following such strategy, a truncated aptamer AP32-4 with the length of only 15 nt was eventually screened. The dissociation constant of 77.22 ± 11.27 nM is almost equivalent to the original AP32. Furthermore, an aptamer-based fluorescent biosensor for metronidazole was constructed based on AP32-4. With the help of exonuclease-assisted target-recycling amplification, the biosensor exhibits a linear detection range of 25-800 nM, and the detection limit of 10.50 nM. The biosensor was applied to detect metronidazole in honey samples. The results show that not only an efficient strategy for screening robust and practicable aptamers, but also an ultrahigh sensitive detection platform for metronidazole were established.

Keywords: Aptamer; Fluorescent biosensor; Magnetic beads SELEX; Metronidazole; Target-recycling signal amplification.