Three-dimensional magnetophoretic bioassay based on artificial intelligence-mediated load assignment for the femtomolar and washing-free detection of analytes

The quantification of low-abundance biomarkers or trace harmful substances in complex samples at femtomolar-level sensitivity requires labor-intensive incubation and multiwashing steps. Herein, we explore a trajectory/accumulation/color three-dimensional microfluidic magnetophoresis immunoassay (TAC-MMI) based on an artificial intelligence (AI)-assisted load assignment strategy, enabling washing-free and femtomolar detection of interleukin-6 in serum samples and chloramphenicol in food samples within 30 min. The biorecognition between targets and biometric molecules (antigens and antibodies) immobilized on magnetic nanoparticles (MNPs) and polystyrene microsphere-horseradish peroxidase (PM-HRP) conjugate induces MNP-target-PM-HRP immunocomplexes with different magnetic contents, where the concentration of targets is transformed into spatial visualization information through magnetophoretic force using a linear microtube array within a microfluidic chip. The visualization information can be enhanced by the HRP-catalyzed color reaction. Trajectory-accumulation-color of PM in each microtube is precisely assigned significance and identified via AI for bioanalysis. TAC-MMI demonstrates high sensitivity (fM level), rapidity (30 min), and ease of use without washing steps. The three-dimensional sensing strategy based on load-assignment improved sensitivity by more than 96-fold compared with the traditional one-dimensional sensing strategy. Compared to chemiluminescence immunoassay (CLIA), TAC-MMI achieved 15-fold improvement in sensitivity in only one-third of the time required for CLIA, suggesting a promising magnetophoretic bioassay for diagnostic technology.