Ultrafast and ultrasensitive electrochemical aptasensor for multiplexed monitoring of hemorrhagic transformation biomarker in acute ischemic stroke.

Hemorrhagic transformation (HT) remains a life-threatening complication after acute ischemic stroke (AIS), yet no active predictive tool is available for early risk assessment. Herein, we develop an integrated triplex electrochemical aptasensing platform for the parallel and simultaneous quantification of three HT-associated biomarkers-matrix metalloproteinase-9 (MMP-9), fibronectin (FN), and plasminogen activator inhibitor-1 (PAI-1). The system enables parallel detection by inserting three independent screen-printed carbon electrodes (SPCEs) into a multi-channel electrochemical workstation, each functionalized with a distinct aptamer, allowing simultaneous quantification of MMP-9, FN, and PAI-1 within 30 min. Each SPCE is modified with a conductive polypyrrole/poly(3,4-ethylenedioxythiophene):polystyrene sulfonate/polyvinyl alcohol hydrogel and gold nanoparticles, followed by immobilization of specific thiolated aptamers via Au-S bonds. Upon target binding, aptamer conformational changes induce a concentration-dependent decrease in differential pulse voltammetry current. The sensor achieves ultralow detection limits of 4.04 fg/mL, 0.30 fg/mL, and 2.79 fg/mL for MMP-9, FN, and PAI-1, respectively, with a total assay time of 30 min. Clinical validation using plasma from 12 AIS patients demonstrates excellent agreement with enzyme-linked immunosorbent assay (mean bias <10%) and high reproducibility (RSD <5%). This work presents the first electrochemical aptasensor for FN and PAI-1 detection and provides a clinically validated, point-of-care-compatible platform for personalized HT risk prediction in thrombolytic therapy.