An Integrated Cartridge-Based Multiplex Chemiluminescent Immunosensor Platform for the Simultaneous Quantification of Five Coagulation and Fibrinolysis Biomarkers

Abstract

This study presents a novel single-integrated cartridge-based multiplex chemiluminescence immunosensor (SIC-MCLIS) platform designed for rapid, simultaneous quantification of five key coagulation and fibrinolysis biomarkers-thrombomodulin (TM), thrombin–antithrombin complex (TAT), plasmin-α2-plasmin inhibitor complex (PIC), tissue plasminogen activator–inhibitor complex (t-PAIC), and D-dimer. This fully automated “sample-in to result-out” system integrates all reagents and consumables within a compact reagent cartridge and analyzer, enabling sensitive detection from only 200 μL of plasma within 25 min. The platform employs a streptavidin–biotin amplification strategy and features precise pipetting, uniform temperature control, efficient washing, and low background signal, ensuring high accuracy and reproducibility. The platform exhibits enhanced analytical performance, with limits of detection (LoD) of 0.5 ng/mL for TAT, 1.0 TU/mL for TM, 0.06 μg/mL for PIC, 0.75 ng/mL for t-PAIC, and 5 ng/mL for D-dimer. The repeatability coefficient of variation (CV) is below 8%, the within-laboratory CV is under 10%, bias remains within 8%, and linearity is demonstrated by an R2 value exceeding 0.99. A method comparison study conducted on 228 clinical samples against the reference system HISCL-5000 showed a strong correlation (R2 > 0.99) and agreement exceeding 96% within the Bland-Altman limits. Overall, this innovative multiplex immunoassay system offers a rapid, cost-effective, and accessible solution for comprehensive coagulation-fibrinolysis assessment, with significant potential to improve real-time clinical management of thrombotic and coagulopathic disorders, especially in emergency and point-of-care settings.