A mechanical buoyant microsphere immunoassay platform for single-embryo culture medium analysis

Immunoassays are indispensable tools in biomedical research and diagnostics, but conventional approaches such as ELISA demand large sample volumes and long assay times, making them unsuitable for precious biological fluids like single-embryo culture medium (ECM). Here we present a novel mechanical buoyant microsphere immunoassay platform that enables reliable analysis of microliter-scale samples. Buoyant hollow glass microspheres (40-60 μm) serve as the reaction carriers, passively aggregating by buoyancy for extraction and sequential transfer across reagent droplets. Through a droplet-based design, in which droplets are stabilized on hydrophilic traps patterned on a hydrophobic surface, each assay requires only 2 μL of sample. By using a custom quantification chip to standardize the microsphere number to 25, the system achieved high reproducibility (coefficients of variation, CV values < 10 %) and enhanced fluorescence signals. The assay demonstrated a limit of detection (LOD) below 0.4 pg/mL for human IL-1β. Importantly, with a wind-assisted mixing module, biomolecular interactions were accelerated and the total assay time was reduced to about 18 min. Validation with single-embryo ECM from a mouse model, followed by embryo transfer and implantation assessment, further demonstrated feasibility for embryo-related biomarker studies. Overall, this platform provides a rapid, sensitive, and automation-ready solution optimized for low-volume samples, with strong potential for non-invasive embryo assessment and broader biomedical applications.