Gold nanoparticle based photometric determination of tobramycin by using new specific DNA aptamers

Abstract

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 (Ks) were determined by a fluorescent method. An aptamer termed "Ap 32", with a K 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 K 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. Graphical abstract A 34-nucleotide aptamer specific for tobramycin was obtained through magnetic beads-based systematic evolution of ligands by exponential enrichment (SELEX) and structural analysis-based rational post-SELEX truncation, and then applied to the determination of tobramycin using a gold nanoparticle-based photometric assay.

Chemical Modification of Aptamers for Increased Binding Affinity in Diagnostic Applications: Current Status and Future Prospects

“…After target binding, the structural conformation of the aptamer changes, which alters the impedance of the electrode and allows detection of the analyte. These aptamer-based biosensors have been used for the detection of glycated hemoglobin (an important diagnostic marker in diabetes) and the antibiotic tobramycin, amongst others [ 54 , 55 ]. Truncated aptamers have also been implemented in an optical biolayer interferometry (BLI)-based aptasensor for the detection of the marine neurotoxin Gonyautoxin 1/4 [ 49 ].…”

Aptamer-Based Biosensors for Antibiotic Detection: A Review

“…Han et al [ 124 ] developed a magnetic bead-based SELEX to identify 37 ssDNA aptamers specific for tobramycin using a fluorescent method based on the reported principle by Ma’s team [ 125 ], in which they developed a colorimetric aptasensor for the determination of tobramycin in milk and chicken eggs based on the adsorption of ssDNA aptamers on the surface of AuNPs. With the sensor of Ma et al [ 125 ] the one reaching lowest LOD was developed ( Table 6 ).…”

Recent Advances in Optical Detection of Aminoglycosides

“…The employment of aptamers not only improves the detection specificity but also improves the detection sensitivity. The aptamer-based colorimetric sensing strategy has been developed for the detection of kanamycin, tobramycin, and neomycin with LODs of 8.1 nM, 23.1 nM, and 27 nM, respectively, in which the ratio A620/A520 was employed as the parameter for quantitative detection [55][56][57]. In order to improve the performance of the Au NP-based colorimetric aptasensor for the detection of antibiotics, Shayesteh's group employed a polyA-tailed aptamer and poly(diallyl dimethyl ammonium chloride) (PDDA) to replace the original aptamer and salt, respectively, and constructed a colorimetric assay for the detection of kanamycin ( Figure 5B).…”