Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

Abstract

This work was the first to report that the kanamycin-binding DNA aptamer (5′-TGG GGG TTG AGG CTA AGC CGA-3′) can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA–TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

Targeting human telomeric G-quadruplex DNA with antitumour natural alkaloid aristololactam-β-D-glucoside and its comparison with daunomycin

“…The extent of TO displacement by the externally added drug can be correlated to its G4‐DNA binding ability, which results a decrease in fluorescent signal of the TO‐G4‐DNA complex with subsequent increase in the drug concentration. The TO displacement by ADG and DAN was quantified by the DC50 values, which represents the concentration of drug needed to displace 50% of thiazole orange . Aristololactam‐β‐D‐glucoside and DAN both quenched the fluorescence intensity of TO‐quadruplex complex considerably, and the DC50 values were calculated to be 1.02 and 0.68 for ADG and DAN, respectively (Figure ).…”

Fluorescent aptasensor for chloramphenicol detection using DIL-encapsulated liposome as nanotracer

“…This is because aptamer has even higher affinity towards targets than antibody (Xiao et al, 2005). Among various species fluorescent bio-probes, small organic molecules, for instance 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanineperchlorate (DIL) (Verbovetski et al, 2002), rhodamine (Johnson et al, 1980), fluorescein (Vermes et al, 1995), are popular because they are rich in variety and easy to use (Xu and Lu, 2009 analyte in aqueous phase (Niu et al, 2009). Accordingly, the development of new types of aqueous and specific fluorescent aptamer probes for antibiotic contaminant is highly desirable.…”

“…To fabricate fluorescence assay forreal-time monitoring of water soluble antibiotic contaminant in food samples, for instance chloramphenicol (CAP), kanamycin (Kana), has attracted more and more attention during these years Liu et al, 2015;Xing et al, 2015;Guo et al, 2015). Because, upon the overdose of CAP intake from food or medicinal, it can result in many serious negative impacts, including bone marrow suppression, gray baby syndrome, kidney damage and allergic reactions (Wang et al, 2010;Yu et al, 2014).…”

Colorimetric detection of kanamycin based on analyte-protected silver nanoparticles and aptamer-selective sensing mechanism

“…However, over-using Kana or excessive 4 consumption of Kana contained animal-derived food may lead to ultimate accumulation in human 5 bodies, which may cause serious side effects including hearing damage, kidney injury and allergic 6 shock [2,3]. To keep these side effects to a minimum, several methods, such as enzyme-linked 7 immunosorbent assay [4,5], capillary electrophoresis [6, 7], high performance liquid 8 chromatography [8,9], surface plasmon resonance [10, 11] and electrochemical methods [12,13] For investigation of selectivity-enhancement effect of K-aptamer, firstly, 25 µL of 0.6 µg 8 ·mL -1 Kana was added into a dispersion of 100 µL unmodified AgNPs and incubated for 5 9 minutes at room temperature. Then, 50 µL of 1.0 µM K-aptamer solution was added into 10 the above Kana-protected AgNPs dispersion and the resulting mixture was allowed to stand 11 by 10 minutes to perform the aptameric binding [25].…”