A magnetic bead-based SELEX was applied to identify 37 single-stranded DNA aptamers specific for tobramycin after ten rounds of selection. The aptamers were classified into nine families according to sequence analysis. Among them, several aptamers with typical sequences were selected and their dissociation constants (Kds) were determined by a fluorescent method. An aptamer termed “Ap 32”, with a Kd value of 56.8 ± 4.6 nM, possesses the highest affinity and satisfactory specificity. Theoretical modeling showed that nucleotides 14–18 and 26–29 play a most significant role in the interaction between aptamer and tobramycin. Subsequently, the sequence of Ap 32 was optimized through rationally designed truncation. The truncated aptamer Ap 32–2 consists of 34 nucleotides and has a Kd that is similar to the original one. It was chosen as the optimal aptamer for use in the assay and was immobilized on gold nanoparticles. On addition of tobramycin, the color turns from red to purple. The findings were used to design a photometric assay (best performed at 520 nm) that has a linear response in the 100 nM to 1.4 μM concentration range, with a 37.9 nM detection limit. The method was successfully applied to the determination of tobramycin in (spiked) honey samples.
Microchimica Acta – Springer Journals
Published: Dec 2, 2017
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