Construction of a ratiometric electrochemical aptasensor based on graphdiyne-methylene blue and Fc-labeled hairpin for cyclic signal amplification detection of kanamycin

Based on ferrocene (Fc)-labeled primers and graphdiyne-methylene blue (GDY-MB) nanocomposite, a ratiometric electrochemical aptasensor was prepared for high-precision detection of kanamycin (KAN). The Fc-labeled primer hybridized with the aptamer forms a rigid structure. When KAN was specifically bound to an aptamer, a primer with a self-complementary sequence was released to form a hairpin structure (HP), drawing the Fc close to the electrode surface, resulting in enhanced IFc. Exonuclease I (Exo I) was used to cleave the single-stranded aptamer and release the target, effectively increasing the cyclic amplification signal. The phosphorylation was carried out at the 3′ end of the primers to prevent HP cleavage. The chitosan - antimony tin oxide (CS-ATO) and AuNPs were used to further modified the electrodes to double the signal amplification. Under the optimal experimental conditions, the KAN concentration exhibited a strong linear relationship with IFc/IMB. The R2 (0.997) of the calibration curve was greater than the R2 (0.960) of the standard curve of KAN concentration and IFc, which indicates the robustness of the ratiometric electrochemical sensor. The limit of detection (LOD) was 6.044 nM. The aptasensor exhibits strong stability and specificity for successfully detection of KAN in real milk samples.