An ultrasensitive homogeneous aptasensor for kanamycin based on upconversion fluorescence resonance energy transfer

• An aptasensor is constructed to detect kanamycin based on upconversion fluorescence resonance energy transfer.
• The conformation change of kanamycin aptamer into a hairpin structure after interacting with the target is accompanied by the alteration of energy-transfer efficiency.
• The aptasensor for kanamycin detection is ultrasensitive.
• The aptasensor is robust in human serum samples indicating its prospect of practical applications.

We developed an ultrasensitive fluorescence resonance energy transfer (FRET) aptasensor for kanamycin detection, using upconversion nanoparticles (UCNPs) as the energy donor and graphene as the energy acceptor. Oleic acid modified upconversion nanoparticles were synthesized through a hydrothermal process followed by a ligand exchange with hexanedioic acid. The kanamycin aptamer (5'-NH2-AGATGGGGGTTGAGGCTAAGCCGA-3') was tagged to UCNPs through an EDC–NHS protocol. The π–π stacking interaction between the aptamer and graphene brought UCNPs and graphene in close proximity and hence initiated the FRET process resulting in quenching of UCNPs fluorescence. The addition of kanamycin to the UCNPs–aptamer–graphene complex caused the fluorescence recovery because of the blocking of the energy transfer, which was induced by the conformation change of aptamer into a hairpin structure. A linear calibration was obtained between the fluorescence intensity and the logarithm of kanamycin concentration in the range from 0.01 nM to 3 nM in aqueous buffer solution, with a detection limit of 9 pM. The aptasensor was also applicable in diluted human serum sample with a linear range from 0.03 nM to 3 nM and a detection limit of 18 pM. The aptasensor showed good specificity towards kanamycin without being disturbed by other antibiotics. The ultrahigh sensitivity and pronounced robustness in complicated sample matrix suggested promising prospect of the aptasensor in practical applications.