Abstract

To enable highly sensitive and automated detection of alpha-fetoprotein (AFP), we developed a capillary-driven microfluidic aptasensor that uniquely integrates label-free DNase I-assisted target recycling with electrochemical redox cycling using ruthenium(III)-methylene blue (Ru-MB) redox species. The aptamer was immobilized on a screen-printed graphene electrode through π-π interactions, enhancing redox electron transfer due to the negative charge of the phosphodiester backbone. Upon binding to AFP, the aptamer dissociated from the electrode, thereby reducing surface negative charges and lowering the current signal in a signal-off manner. DNase I further cleaved the dissociated aptamer, freeing AFP for multiple binding cycles, which enhanced signal differentiation. Current response changes from the redox probe indicator in the AFP sample were measured using chronoamperometry, with the AFP concentration calculated from the current change, ∆I. The system was integrated into a capillary-driven microfluidic device for automated enzyme signal amplification and sequential reagent delivery, requiring only two pipetting steps: adding the sample and the buffer solution. The device showed a linear detection range of 0.5–500 ng/mL and a detection limit of 0.5 ng/mL, demonstrating its potential for early diagnosis of hepatocellular carcinoma (HCC) while maintaining excellent specificity for samples from both healthy individuals and HCC patients.