A highly sensitive ratiometric electrochemical aptasensor for prostate specific antigen detection based on toehold-mediated strand displacement reaction

In this work, a novel ratiometric electrochemical aptasensor was designed for prostate specific antigen (PSA) detection based on toehold-mediated strand displacement reaction (TMSDR). For PSA detection, PSA could bind with the aptamer sequence in hairpin probe 1 (HP1), and HP1/PSA complex was obtained. Then, in the presence of S3 DNA, HP1/PSA could trigger two TMSDR processes by hybridizing with Fc-S2/S1 double-stranded DNA, which would generate a large amount of Fc-S2 and recycle HP1/PSA. This enabled the hybridization of Fc-S2 with methylene blue labeled thiolated hairpin probe 2 (MB-HP2) immobilized on the electrode surface, causing MB molecules away from the electrode and Fc close to the electrode. Thus, with the increasing PSA concentrations, the current of MB (I_MB) decreased and the current of Fc (I_Fc) increased. The ratio of I_Fc/I_MB was shown to be linear with the logarithm of PSA concentration from 1 pg mL⁻¹ to 100 ng mL⁻¹, with a detection limit of 0.28 pg mL⁻¹. In addition, the aptasensor had good specificity, reproducibility, repeatability, and stability for PSA detection, and was successfully applied to spiked diluted human serum samples.