PolyA-Bridged Aptamer for Label-Free Electrochemical Aptasensors: Comparable Performance to Thiol-Terminated Aptamer but with Sharply Reduced Cost

Thiol-terminated aptamers (SH-Apt) enable Au-S self-assembly on Au electrodes but face great challenges due to expensive thiol modification and laborious disulfide cleavage steps. Poly adenine (PolyA)-mediated aptamers offer a simpler and cost-effective alternative, yet lack systematic comparative studies against Au-S immobilization for critical metrics in electrochemical aptasensors. In this work, we present a systematic comparative evaluation of PolyA-bridged aptamer (PolyA-Apt) and SH-Apt immobilization strategies on Au electrodes for electrochemical aptasensor, using metronidazole (MTZ) as a model target and electrochemical impedance spectroscopy (EIS) for performance assessment. The experimental results demonstrated that the PolyA-Apt modified Au electrode (#Au-PolyA-Apt) achieved analytical performance comparable to thiol-based counterparts (SH-Apt) for MTZ detection, matching its linear range and yielding a near-equivalent limit of detection (7.8 vs 3.4 pM), yet offering a remarkable 74-fold decrease in aptasensor fabrication cost. Furthermore, the developed #Au-PolyA-Apt aptasensor demonstrated excellent selectivity against interfering antibiotics, good storage stability, retaining 91.3% of its initial response after 10 days of storage, and high intra/inter-batch reproducibility. Successful determination of MTZ in honey and tapping water validated its real-world applicability, directly bridging critical knowledge gaps in fabrication cost, sensitivity, and scalability to advance electrochemical aptasensing toward commercial viability.