Singlicate bead-based immunoassay for biomarker quantitation in sub-microliter volume sample

Point-of-care testing (PoCT) devices are becoming increasingly essential for widespread disease diagnostics. Hence, it becomes necessary to develop solutions for detecting biomarkers from minimal-volume samples while remaining accessible and user-friendly. This study presents a new method called FoCUS (Forward scattering Correction Using a Single-bead). FoCUS combines tiny beads floating in liquid with a small chip to measure very small amounts of sample. It can analyze a single drop using just one bead. The platform employs a novel signal correction strategy to enhance fluorescence signal sensitivity using forward scattering signals obtained simultaneously. Compared to conventional methods, the FoCUS platform achieves a 1000-fold reduction in the required sample volume, a 2.7-fold decrease in the relative standard deviation of sample signals, and a 4.3-fold improvement in detection limits. The platform successfully detected relevant myoglobin levels from 0.1 μL of sample, demonstrating its potential for multiplex biomarker immunoassays in point-of-care applications where sample volumes are limited.