A one-for-four redox probe strategy for multiplex electrochemical POCT of thrombotic biomarkers

Thrombotic diseases remain a major global health concern with high morbidity and mortality. Four biomarkers (TAT, t-PAIC, TM, PIC) enable real-time monitoring of coagulation, addressing limitations of traditional diagnostics. Herein, we developed a multiplex electrochemical immunosensor for the simultaneous detection of these biomarkers. The sensing platform is constructed on a glassy carbon electrode modified with gold nanoparticles and laser-induced graphene (AuNPs-LIG), which enhances conductivity and signal output. A "One-for-Four" strategy was employed using four distinct redox probes (AQ, OAP, MB, PPD), each conjugated to a specific antibody via amidation and anchored to functionalized carbon nanotubes (Au@CNTs), forming a multichannel signal amplification system with high sensitivity. The immunosensor exhibited excellent linearity (10-100 ng/mL, R² > 0.98) and low detection limits: 3.99 ng/mL (TAT), 5.25 ng/mL (t-PAIC), 2.51 ng/mL (TM), and 4.20 ng/mL (PIC). It also demonstrated high selectivity, reproducibility and long-term stability. Recovery in human plasma ranged from 84.1% to 112.21%, indicating strong applicability to complex clinical samples. This work presents a robust, portable POCT platform with great potential for early diagnosis and dynamic monitoring of thrombotic diseases.