An on-axis integrated microfluidic chemiluminescence immunoassay platform for rapid multiplex biomarker quantification

Multiplexed biomarker quantification from a single specimen remains challenging because it requires aliquoting, target capture, washing, and signal readout within a compact assay format. In microfluidic chemiluminescence immunoassay (CLIA) implementations, these operations are often distributed across separate steps and locations, resulting in repeated handling, transfer-related variability, and an increased risk of carryover. Here we report a two-station microfluidic CLIA analyzer coupled with a reagent-preloaded triplex disc that re-engineers the workflow through an on-axis integrated architecture. In this work, on-axis integration denotes coaxial co-localization of disc actuation, temperature regulation, magnetic bead manipulation, and photomultiplier tube (PMT)-based chemiluminescence readout within a single axial workspace, rather than merely rotating a disc around its center. This arrangement enables a self-contained in situ reaction and in situ readout sequence. A disposable, reagent-preloaded microfluidic disc and tubing-free operation streamline handling and reduce transfer-related variability and carryover. Using cardiac troponin I (cTnI), creatine kinase-MB (CK-MB), and myoglobin (MYO) as model analytes, the triplex measurement for one sample was completed within 10 min. The platform exhibited linear responses over concentration ranges of 0.0015–30 ng/mL (cTnI), 0.1–300 ng/mL (CK-MB), and 1–4000 ng/mL (MYO), with limits of detection of 0.001 ng/mL, 0.1 ng/mL, and 1 ng/mL, respectively. The assay maintained robust specificity, precision, accuracy, and tolerance to common interferents. Method comparison involving 255 clinical serum specimens showed strong agreement between the proposed platform and a reference automated clinical CLIA analyzer (R² > 0.97 for all three biomarkers). This work highlights an integration strategy that compresses a multi-step CLIA workflow into a compact, mechanically simplified microfluidic system for rapid multiplexed chemiluminescence testing.