Electrochemical-LSPR Multiplexed Immunoassay with a Multi-Analyte Index for Sepsis-Associated Biomarkers

Multiplexed immunoassays frequently face challenges associated with signal cross-reactivity, matrix effects, and the integration of analytes with disparate concentration ranges and transduction efficiencies. Here, we report an electrochemical multiplexed immunoassay implemented on a four-working-electrode screen-printed carbon electrode (4-WE SPCE) platform, which was complemented with localized surface plasmon resonance (LSPR). Distinct redox probesmethylene blue (MB), cysteine (Cys), and ruthenium hexamine chloride (RuHex) were assigned to individual working electrodes, enabling parallel differential pulse voltammetry (DPV) measurements of three inflammatory biomarkers in phosphate-buffered saline (PBS) and 50% human serum. Blank samples were analyzed separately, and the nonzero calibration ranges were 0.5-20 ng mL^-1 for procalcitonin (PCT) and 0.5-20 μg mL^-1 for both C-reactive protein (CRP) and serum amyloid A (SAA). The bioassay demonstrated concentration-dependent current suppression, with calculated limits of detection (LoDs) of 0.0255 ng mL^-1 for PCT, 1.60 μg mL^-1 for CRP, and 0.0670 μg mL^-1 for SAA in PBS, and 6.99 ng mL^-1 for PCT, 0.0556 μg mL^-1 for CRP, and 0.193 μg mL^-1 for SAA in 50% human serum. Minimal cross-reactivity between biomarkers was observed, supporting analytical selectivity, while the measurements showed repeatability across replicate experiments (n ≥ 3). Gold nanoparticle (AuNP) surface engineering enhanced electron-transfer kinetics and antibody immobilization, producing analyte-specific electrochemical signatures. Complementary LSPR measurements provided an orthogonal optical readout that offered evidence of antibody-antigen binding at the plasmonic interface, strengthening confidence in assay specificity and performance. To support multiplexed signal integration, a dimensionless multianalyte index (MAI) based on normalized concentrations was introduced as a proof-of-concept analytical framework for combining heterogeneous biomarker signals. This MAI framework enables standardized interpretation of multiplexed electrochemical measurements using sepsis-associated inflammatory biomarkers, highlighting its potential applicability to broader multiplexed immunoassay platforms.