Development of new aptamers against antibiotics and detection system using aptamers

Abstract

Currently, the use of antibiotics as prophylactic and therapeutic agents for microbial infections is increasing rapidly. This circumstance results in side-effects that range from mild to very serious with chemical poisoning and allergic reactions depending on the transfer of the residues in the food chain into humans. In this study, several antibiotic-specific aptamers were selected and used to propose an easier, more reliable, and more accurate sensing system for each antibiotic to monitor the antibiotic residues in the pharmaceutical preparations and food products. At first, a gold nanoparticle (AuNP)-based colorimetric aptasensor was introduced to detect kanamycin. A selective kanamycin-binding ssDNA aptamer (Ky2

‘TGGGGGTTGAGGCTAAGCCGA’, Kd=78.8 nM) was discovered through the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method using affinity chromatography. The selected aptamer has a high affinity for kanamycin and for kanamycin derivatives such as kanamycin B and tobramycin. Using this aptamer, kanamycin was detected at concentrations as low as 25 nM by the AuNP-based colorimetric method. Because the devised colorimetric method is simple and visible to the naked eye, it has advantages that make it useful for the detection of kanamycin. Furthermore, the newly selected aptamer has many potential applications as a bio-probe for the detection of kanamycin, kanamycin B, and tobramycin in food products.Second, a gold nanoparticle-based dual fluorescence-colorimetric dual method was established as an aptasensor to detect ampicillin using its ssDNA aptamer (AMP17

‘GCGGGCGGTTGTATAGCGG’, Kd=13.4 nM), which was discovered by a magnetic bead-based SELEX technique. The 5’-fluorescein amidite (FAM)-modified aptamer was used as a dual probe to observe fluorescence differences and color changes simultaneously. The lower limits of detection for this dual method were 2 ng/mL by fluorescence and 10 ng/mL by colorimetry for ampicillin in milk. Because these detection limits were below the maximum residue limit of ampicillin, this aptasensor is sensitive enough to detect antibiotics in food products such as milk and animal tissues. In addition, this dual method is more accurate than the methods currently used to detect antibiotics in food products because two detection methods are concurrently applied: fluorescence and colorimetry.Finally, the polymer-based aptasensor, which consists of a fluorescein amidite (FAM)-modified aptamer and coordination polymer nanobelts (CPNBs), was developed by utilizing fluorescence quenching effect to detect sulfadimethoxine residue in food products. The ssDNA aptamer (Su13

‘GAGGGCAACGAGTGTTTATAGA’, Kd=84 nM) for sulfadimethoxine was discovered by a magnetic bead-based SELEX technique, and the CPNBs were produced as a fluorescent quencher by mixing AgNO3 and 4, 4’-bipyridine. The proposed method was able to easily and sensitively detect sulfadimethoxine with a limit of detection (LOD) of 10 ng/mL. Furthermore, the sulfadimethoxine dissolved in real milk was also effectively detected with the same LOD. In addition, using the aptamer as the probe offered a high specificity for sulfadimethoxine over other antibiotics. These results provide ample evidence that the CPNBs-based aptasensor can be used to test for sulfadimethoxine residues in food products.