Chip-Integrated Optical Modulation Biosensing with Microfluidic Background Reduction for High-Sensitivity Fluorescence Immunoassays

Microfluidic fluorescence immunoassays enable sensitive biomarker detection, but performance is limited by inefficient capture on planar surfaces and background fluorescence from unbound labels. Here, we present chip-based optical modulation biosensing (Chip-OMB), combining three-dimensional magnetic bead-based target capture, bead immobilization, internal fluid flushing, and modulated optical background subtraction. Magnetic beads are concentrated and immobilized in the chamber, while controlled on-chip flushing displaces unbound fluorescent labels without requiring user-performed washing after chip loading. Alternating optical interrogation of the bead cluster and surrounding solution enables background subtraction. Using fluorescently labeled magnetic beads, Chip-OMB achieved limits of detection of 0.1 pM in buffer and 22 pM in undiluted plasma, corresponding to 580-fold and 22-fold lower equivalent fluorophore-concentration limits of detection, respectively, than conventional steady-state bulk fluorescence measurements using a plate reader. Measurements correlated with the laboratory optical modulation biosensing system (R2 = 0.98). In an anti-dengue virus serotype 2 (DENV-2) nonstructural protein 1 (NS1) immunoglobulin G (IgG) immunoassay, Chip-OMB achieved a 3 ng/mL limit of detection with a ~4-log dynamic range. Among 56 sera, 29 DENV-2 cases confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) were Panbio IgG-positive and above the exploratory Chip-OMB cutoff, while all 27 study-defined negative controls were below it.