Ultrasensitive and label-free electrochemical aptasensor of kanamycin coupling with hybridization chain reaction and strand-displacement amplification.

This work reports the proof-of-concept of an ultrasensitive label-free electrochemical aptasensor for Kanamycin (Kana) detection coupling strand-displacement amplification (SDA) with hybridization chain reaction (HCR). In the presence of target Kana, the analyte triggers conformational change of hairpin HP1 (HP1) and two-staged SDA to produce short single-stranded DNA (S1) with the aid of KF polymerase and nicking endonuclease. Meanwhile, the as-produced S1 hybridizes with the immobilized hairpin HP2 (HP2) on the electrode to open the hairpin, thereby resulting in the formation of DNA duplex. Thereafter, DNA duplex is selectively digested by Exo III accompanying S1 recycling. The residual single-stranded probe (S2) on the electrode opens another two hairpins in sequence and propagates a chain reaction of hybridization events between two alternating hairpins (H1 and H2) to form a long nicked double-helix. Upon addition of redox-active methylene blue (MB), numerous indicators are intercalated into the grooves of double-helix DNA polymers, each of which produces an electrochemical signal within the applied potentials. Under optimum conditions, the SDA/HCR-based electrochemical aptasensor exhibits a high sensitivity for detection of Kana down to 36 fM with a linear range from 0.05 to 200 pM. Additionally, the as-prepared aptasensor is successfully employed to determinate the Kana in animal derived food (milk). With the advantages of high sensitivity, label-free strategy and excellent selectivity, the developed aptasensor possesses great potential application value in food-safety analysis field.