A highly sensitive, simple and label-free fluorescent aptasensor for tobramycin sensing based on PicoGreen intercalation into DNA duplex regions of three-way junction origami

Abstract Structure-selective light-up dyes are ideal materials for designing label-free DNA-based biosensors and have gained more interest with researchers. In this study, PicoGreen (PG), as a cyanine fluorogenic dye with a low intrinsic fluorescence emission rate, was employed in a design of an aptasensor based on the formation of three-way junction (3WJ) structure to sense tobramycin (TOB), a member of aminoglycoside antibiotic family with a narrow therapeutic index, in a highly sensitive and simple procedure. In fact, the presence of TOB in the assay changes the number of double-stranded sequences in the engineered design, providing the basis for the difference in the intensity of PG fluorescence signal. The designed biosensor showed a wide linear range from 80 nM to 2 µM with a limit of detection (LOD) equal to 21.86 nM in water. Moreover, it was able to determine TOB in human serum, as a complex matrix, in two linear zones of 5–20 µM and 20–300 µM with a LOD of 321.2 nM, with no need for any complicated sample processing, including addition of organic solvents or ultrafiltering, and only through dilution for 40 times with water. Our results indicated that the prepared sensing strategy could be applicable for clinical samples.