Microfluidic Magnetic Analyte Delivery Technique for Separation, Enrichment, and Fluorescence Detection of Ultratrace Biomarkers

A microfluidic magnetic analyte delivery (μMAD) technique was developed to realize sample preparation and ultrasensitive biomarker detection. A simply designed microfluidic device was employed to carry out this technique, including a poly(dimethylsiloxane)-glass hybrid microchip having four straight rectangular channels and a permanent magnet. In the μMAD process, functionalized magnetic beads (MBs) were used to recognize and isolate analytes from a complex sample matrix, deliver analytes into tiny microchannels, and preconcentrate analytes in the magnetic trapping/detection region for in situ fluorescence detection. In the feasibility study and sensitivity optimization, horseradish peroxidase-labeled MBs were used, and critical parameters for the signal amplification performance of μMAD were carefully evaluated. At optimized conditions, a sensitivity improvement of at least 2 orders of magnitude was achieved. As a proof of concept, μMAD was combined with the enzyme-linked immunosorbent assay (ELISA), while carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), and interleukin 6 (IL-6) were selected as model biomarkers. The limits of detection (LODs) of μMAD-ELISA were as low as 0.29 pg/mL for CEA, 0.047 pg/mL for PSA, and 0.021 pg/mL for IL-6, which corresponded to an over 200-fold reduction compared to their commercial ELISA results. Meanwhile, μMAD-ELISA revealed high selectivity and reproducibility. In clinical sample analysis, good accuracy was acquired for human serum analysis relative to commercial ELISA kits, and satisfied recoveries of 85.1-102% with RSDs of 1.7-9.8% for IL-6 and 84.7-113% with RSDs of 3.2-8.3% for interferon-γ were obtained. This ultrasensitive and easy operation technique provides a valuable approach for trace-level biomarker detection for practical applications.