A digital fluorescence immunoassay platform using a self-driven microfluidic cartridge with magnetic capture

Self-driven microfluidic devices have significantly advanced point-of-care testing (POCT) by enabling integration and automation of all the components required for biochemical analysis through capillary-driven fluid flow, eliminating the need for external pumps. However, these systems often face challenges such as slow molecular diffusion and complex designs that require external actuation to enhance immunoreactions. In this study, we present a fully integrated self-driven microfluidic fluorescence immunoassay cartridge (SDMFIC) for precise quantification, employing magnetic nanoparticles (MNPs) as a movable substrate and quantum dots (QDs) as a fluorescent tag. The SDMFIC features an inlet zone for sample loading, a detection probe zone loaded with QDs conjugated with C-reactive protein (CRP) antibodies, a capturing probe zone loaded with MNPs conjugated with capturing antibodies and a sensing zone embedded with a hard magnet for immunocomplex capture. Additionally, a blister filled with PBS integrated into SDMFIC enables manual actuation for removing unbound components ensuring accurate quantification using a portable digital fluorescence imaging system. The SDMFIC demonstrated robust analytical performance in both saliva and serum with detection limits of 17 pg/mL and 20 pg/mL, respectively, and a dynamic range spanning five orders of magnitude (0.01-200 ng/mL). Machine learning models were employed to predict and classify various CRP concentrations, achieving 100 % accuracy in saliva and 95.24 % in serum, thereby demonstrating its efficacy for reliable and precise CRP quantification.