Size/Color Dual-Encoded Electrochemiluminescence for Multiplexed Immunoassay of Cardiac Biomarkers

Abstract Electrochemiluminescence immunoassay (ECLIA) is a powerful analytical method for protein biomarker detection, and plays a critical role in early disease diagnosis and clinical monitoring. However, conventional ECLIA is limited by poor multiplexing capacity, typically allowing only single-target measurement per run. Here, we report a dual-encoded strategy for ECL signal resolution that enables simultaneous determination of multiple biomarkers from a single sample. Four types of magnetic microbeads (MMBs), distinguished by both diameter and fluorescent dye labeling, were employed as solid carriers in a multiplexed sandwich immunoassay format. Three of the bead types have a diameter of 3 μm, while the fourth measures 1.5 μm, allowing the latter to be readily identified by bright-field (BF) imaging based on size. The remaining three 3 μm beads are further differentiated by their fluorescence (FL) colors. By combining this size/color dual-encoding scheme with ECL imaging technology, multiplexed recognition and quantification of biomarkers is achieved. The method was validated by simultaneous detection of four cardiac biomarkers, namely cardiac troponin I (cTnI), C-reactive protein (CRP), D-dimer, and myoglobin (Myo). The multiplexed ECLIA demonstrated a wide linear range spanning to 3 orders of magnitude, with detection limits of 0.0044, 0.55, 0.99, 0.30 ng mL−1 for cTnI, CRP, D-dimer, and Myo, respectively. This work offers a facile route to construct multiplexed ECLIA platforms and holds promise for further enhancement of detection throughput through combined encoding strategies.