Enhanced sensitivity in acute myocardial infarction diagnosis through optimized multiplex biomarker detection on a paper-based microfluidic platform with individual control capabilities

Acute myocardial infarction (AMI) is a severe condition with high mortality, requiring timely diagnosis, especially in low-resource settings where centralized testing is unavailable. Leveraging our previously developed, reliable, highly integrated on-chip rotary valving system on the microfluidic paper-based analytical device (µPAD), we propose a novel multiplex paper-based microfluidics immunoassay platform for the onsite detection of key cardiac biomarkers—Myoglobin (Myo), cardiac troponin I (cTnI), and creatine kinase-MB (CKMB)—in artificial serum. It enhances assay performance through individually controlled assay conditions, facilitating rapid AMI diagnosis. Compared to existing paper-based multiplex microfluidic ELISA systems, our µPAD uniquely enables the assay of each biomarker under separate, optimally controlled incubation conditions within a single test through the valving system, which leads to optimized signal-to-noise ratio (SNR) performance and introduces more flexibility in multiplex testing. The valving system in the µPAD is driven by a single servo motor, which is controlled via a microcontroller and an interactive touchscreen. These components are integrated into a highly programmable, easy-to-assemble platform that offers fast, low-cost, and user-friendly operation. Utilizing our platform, autonomous ELISA assays were performed on Myo, cTnI, and CKMB for calibration, achieving limits of detection (LOD) of 1.43 ng/mL for Myo, 0.2 ng/mL for cTnI, and 0.735 ng/mL for CKMB. Subsequent spike-and-recovery experiments also confirmed the platform's accuracy and precision in obtaining the test results within 30 minutes and 20 μL serum sample. These results highlight the platform's transformative potential for clinical AMI diagnosis and commercialization, providing a cost-effective and accessible solution for point-of-care testing in diverse settings.