Development of Structure-Switching Aptamers for Kanamycin Detection Based on Fluorescence Resonance Energy Transfer.

Xinyue Ma,Hongjing Sun,Ruifang Su,Mingdi Zhang,Shangna Qiao,Chunyan Sun

doi:10.3389/fchem.2019.00029

Abstract

The structure-switching aptamers are designed for the simple and rapid detection of kanamycin based on the signal transduction principle of fluorescence resonance energy transfer (FRET). The structure switch is composed of kanamycin-binding aptamers and the complementary strands, respectively labeled with fluorophore and quencher, denoted as FDNA and QDNA. In the absence of kanamycin, FDNA and QDNA form the double helix structure through the complementary pairing of bases. The fluorophore and the quencher are brought into close proximity, which results in the fluorescence quenching because of the FRET mechanism. In the presence of kanamycin, the FDNA specifically bind to the target due to the high affinity of aptamers, and the QDNA are dissociated. The specific recognition between aptamers and kanamycin will obstruct the formation of structure switch and reduce the efficiency of FRET between FDNA and QDNA, thus leading to the fluorescence enhancement. Therefore, based on the structure-switching aptamers, a simple fluorescent assay for rapid detection of kanamycin was developed. Under optimal conditions, there was a good linear relationship between kanamycin concentration and the fluorescence signal recovery. The linear range of this method in milk samples was 100–600 nM with the detection limit of 13.52 nM (3σ), which is well below the maximum residue limit (MRL) of kanamycin in milk. This method shows excellent selectivity for kanamycin over the other common antibiotics. The structure-switching aptamers have been successfully applied to the detection of kanamycin spiked in milk samples with the satisfying recoveries between 101.3 and 109.1%, which is well-consistent with the results from LC-MS/MS. Due to the outstanding advantages of facile operation, rapid detection, high sensitivity, excellent specificity, and low cost, the application and extension of this strategy for rapid determination of antibiotics in food samples may greatly improve the efficiency in food safety and quality supervision.

Highlights

Kanamycin is an aminoglycoside antibiotic purified from Streptomyces kanamyceticus
This structure switch was composed of a FAM fluorophore-labeled kanamycin-binding aptamer (FDNA) and a short oligonucleotide (QDNA) modified with a Dabcyl quencher
When the time reached 50 min, the reaction temperature was lowered to 20◦C, free QDNA recombined with free FDNA to form a stable structure switch, the fluorescence intensity dropped

Summary

Introduction

Kanamycin is an aminoglycoside antibiotic purified from Streptomyces kanamyceticus. Kanamycin is widely used to treat gram-negative and gram-positive infectious diseases in humans and veterinarians. The analytical methods for kanamycin mainly include instrumental analysis, immunoassay and biosensor, such as capillary electrophoresis (Kaale et al, 2003; El-Attug et al, 2011), high performance liquid chromatography (HPLC) (Chen et al, 2006; Blanchaert et al, 2013), enzymelinked immune sorbent assay (ELISA) (Loomans et al, 2003), surface plasmon resonance (SPR) (Raz et al, 2009; Frasconi et al, 2010), and electrochemical immunosensor (Wei et al, 2012), optical aptasensor (Ha et al, 2017; Dehghani et al, 2018; Liu et al, 2018; Tang et al, 2018; Zhu et al, 2018), electrochemistry aptasensor (Sharma et al, 2017; Li et al, 2018), etc. It is very meaningful to establish a convenient, sensitive and low-cost method for rapid and efficient detection of kanamycin in complex food matrix

Methods

Results

Conclusion