Abstract
An electrochemical aptamer-based (E-AB) sensor for myeloperoxidase (MPO) was developed that combined a gold nanoparticle /MXene scaffold with freeze-thaw-directed aptamer immobilization and probe-density optimization. Unlike using nanocomposite amplification alone, this strategy regulates probe density and accessibility at the bioreceptor layer to improve target-coupled signal transduction. Using the same aptamer solution, the freeze-thaw method yielded 60.77% greater MPO-induced signal suppression and a 2.4-fold lower regression-derived limit of detection (LOD) than conventional incubation. After target incubation for 50 min at 4 °C, the sensor showed a linear response from 5.00 to 250.0 ng/mL in 1% plasma matrix (equivalent to 500 ~ 25,000 ng/mL in undiluted plasma), with an LOD of 4.15 ng/mL (equivalent to 415 ng/mL in undiluted plasma). These results illustrated that coupling conductive nanointerface design with aptamer-layer organization might provide a practical route for antibody-free and H2O2-independent MPO determination.