Integrated 3D-Printed Microfluidic Device for Immunocapture and Electrochemical Assessment of Transferrin Saturation in Point-of-Care Stroke Diagnostics

: A 3D-printed electrochemical microfluidic device (3D-EMD) was developed to assess the transferrin saturation (TSAT) biomarker in ischemic stroke patients. The all-in-one 3D-EMD integrates a strategically engineered immunoassay module for the direct and selective isolation of transferrin (Tf) from unpretreated clinical samples, unaffected by sample coloration, with an interchangeable electrochemical sensor for the simultaneous detection of Tf and Tf-bound iron. Both modules are interconnected through microfluidic channels whose flow is regulated by a cylindrical rotary valve. The analytical workflow enables magnetic bead-based direct Tf immunocapture and simultaneous electrochemical detection of Tf and Tf-bound iron via square wave voltammetry, allowing TSAT assessment within 60 min using only 50 μL of sample. Validation with certified reference materials demonstrated excellent accuracy (Er ≤ 5%) and precision (RSD ≤ 4%). Application to human serum from ischemic stroke patients showed strong correlation (r = 0.87) and agreement (slope 0.9 ± 0.3; intercept 6 ± 10; p < 0.05) with the urea-PAGE reference method, which typically requires up to 18 h. Overall, the 3D-EMD constitutes an elegant, fully integrated dual-functionality platform that seamlessly combines customizable sample preparation with online electrochemical detection in a single device. This configuration enables direct serum analysis and supports clinical decision-making in time-critical conditions. The device shows strong potential as a rapid point-of-care testing candidate for ischemic stroke and as a next-generation platform for broader clinical diagnostics.