As antibiotic resistance has risen as one of the major
health concerns
associated with infectious diseases due to the reduced efficacy of
antibiotics, rapid and sensitive detection of antibiotic resistance
genes is critical for more effective and faster treatment of infectious
diseases. A class of programmable DNA-binding domains called transcriptional
activator-like effectors (TALEs) provides a novel scaffold for designing
versatile DNA-binding proteins due to their modularity and predictability.
Here, we developed a simple, rapid, and sensitive system for detecting
antibiotic resistance genes by exploring the potential of TALE proteins
for the creation of a sequence-specific DNA diagnostic along with
2D-nanosheet graphene oxide (GO). TALEs were engineered to directly
recognize the specific double-stranded (ds) DNA sequences present
in the tetracycline resistance gene (tetM), avoiding
the need for dsDNA denaturation and renaturation. We take advantage
of the GO as an effective signal quencher to quantum dot (QD)-labeled
TALEs for creating a turn-on strategy. QD-labeled TALEs are adsorbed
on the GO surface, which will bring QDs in close proximity to GO.
Due to the fluorescence quenching ability of GO, QDs are expected
to be quenched by GO via fluorescence resonance energy transfer (FRET).
QD-labeled TALE binding to the target dsDNA would lead to the conformational
change, which would result in dissociation from the GO surface, thereby
restoring the fluorescence signal. Our sensing system was able to
detect low concentrations of dsDNA sequences in thetetMgene after only 10-minute incubation with the DNA, providing a limit
of detection as low as 1 fM ofStaphylococcus aureusgenomic DNA. This study demonstrated that our approach of utilizing
TALEs as a new diagnostic probe along with GO as a sensing platform
can provide a highly sensitive and rapid method for direct detection
of the antibiotic resistance gene without requiring DNA amplification
or labeling.