Chopped optical biosensing enables temporal background subtraction for high-sensitivity fluorescence immunoassays.

Fluorescence-based immunoassays are widely used for sensitive biomarker detection, but their performance at low analyte concentrations is often limited by background signals originating from unbound fluorophores, Raman scattering, and sample autofluorescence. Here, we introduce chopped optical biosensing (COB), an analytical framework that improves fluorescence immunoassay sensitivity by temporally separating background and target fluorescence. In COB, fluorophore-labeled targets captured on magnetic beads are concentrated into a small detection region using a permanent magnet. An optical chopper periodically gates the excitation beam, enabling fluorescence measurements before bead aggregation (background only) and after aggregation (target + background). Subtraction of these temporally separated signals isolates fluorescence from bead-bound targets while suppressing background contributions from unbound fluorophores, Raman scattering, and sample autofluorescence. By limiting continuous illumination, this approach also reduces photobleaching and stabilizes excitation intensity, while eliminating the need for washing steps or dynamic optical modulation. Analytical evaluation demonstrated detection limits of 61 fM for Atto 532 dye, 0.05 ng/L for interleukin-8 (IL-8), and 3 ng/mL for anti-dengue virus serotype 2 non-structural protein 1 (anti-DENV-2 NS1) immunoglobulin G (IgG), with reproducibility below 15%. The total assay time is approximately 90 min. In a targeted feasibility assessment using 64 well-characterized serum samples, the COB-based anti-DENV-2 NS1 IgG assay correctly classified all samples when compared with reference ELISA results. Combining low complexity, compact design, and energy-efficient operation with high analytical and clinical performance, COB provides a practical foundation for streamlined fluorescence immunoassay workflows and is well suited for future portable diagnostic applications.