Electrochemiluminescence-based multiplex immunoassays for multianalyte bioanalysis: Signal encoding strategies and analytical perspectives

Electrochemiluminescence (ECL) based immunoassays have become important bioanalytical platforms owing to their high sensitivity, low optical background, and intrinsic electrochemical addressability. Moreover, the growing demand for simultaneous detection of multiple biomarkers has driven the rapid development of ECL based multiplex immunoassays (MIAs), in which rational signal encoding and decoding strategies are essential for reliable multianalyte discrimination. Effective multiplexing not only increases the number of detectable analytes, but more importantly requires the generation of analytically orthogonal ECL signals that can be independently resolved within a single assay system. This review provides a strategy-oriented overview of advances in electrochemiluminescence-based multiplex immunoassays for multianalyte bioanalysis, with a particular focus on signal encoding strategies and their analytical implications. Major multiplexing approaches are systematically categorized and discussed, including spectrally resolved, potential-resolved, spatially resolved, and imaging-based ECL strategies. For each category, the underlying encoding principles, representative implementations, and key analytical strengths and limitations are critically examined from an analytical perspective, with emphasis on signal orthogonality, quantitative robustness, and practical applicability. Finally, current challenges and future perspectives are outlined to guide the rational design of reliable ECL multiplex immunoassays for complex bioanalytical applications. • Electrochemiluminescence-based multiplex immunoassays are systematically analyzed from a signal-encoding perspective. • Spectrally, potential, spatially, and imaging encoding strategies are compared with respect to analytical orthogonality and robustness. • The intrinsic constraints of different encoding dimensions on multiplexing capacity are critically discussed. • A unified analytical framework is proposed to guide the rational design of reliable ECL multiplex immunoassays.