An automated, digital immunoassay on a microfluidic cartridge for on-demand cytokine profiling

Patients with critical illness often exhibit profound biological heterogeneity, complicating the identification of effective interventions. Resolving distinct molecular profiles to enable timely treatment decisions remains challenging, as integrating biomarker-guided care into routine monitoring is often hindered by fragmented, batch-based workflows. These manual operations decouple molecular data from the acute clinical timeline and are a barrier to reliable, real-time, near to patient, multi-center implementation. To address this gap, we developed an integrated microfluidic digital immunoassay system that achieves high analytical fidelity through a fully automated, simple workflow. The system utilizes a monolithic disposable cartridge to automate bead-based analyte capture, oil-phase partitioning, and signal amplification, eliminating the manual handling and emulsion steps that typically compromise digital assay robustness. The platform enables protein measurement within 45 minutes, achieving sub-picogram limit of detection ( < 0.13 pg/mL), a dynamic range spanning three orders of magnitude, and strong analytical reproducibility. We demonstrate the clinical utility of the system by profiling a validated panel of inflammatory biomarkers in plasma from critically ill pediatric patients, using low sample volumes. Results show strong agreement with gold-standard multiplex assays (R² = 0.925-0.979). By providing a scalable framework for high-fidelity molecular profiling, this system supports the broader goal of accessible, multi-center biomarker validation and the practical implementation of precision medicine in critical care.