Comparative Evaluation of Electrochemical and Fluorescent Readouts for Immunomagnetically Captured Waterborne Pathogens Using a Biochip with Bifunctional Magnetic Nanoparticles

Detection of waterborne pathogens benefits from measurement strategies that combine sensitivity with a practical workflow. We report a side-by-side metrological comparison of two orthogonal readouts measured on the same immunomagnetically captured cells using bifunctional Fe3O4 nanoparticles (MNPs) conjugated to anti-E. coli IgG and ferrocene (Fc). Under our protocol, these Fc-IgG MNPs achieved capturing efficiency up to 95% for Escherichia coli (E. coli) K12. The electrochemical readout, performed by a microfabricated chip (via differential pulse voltammetry of Fc) shows concentration-dependent signal suppression upon capture of E. coli K12, with an apparent detection limit as low as 10 cells·mL–1 and a broad dynamic range spanning 101 to 109 cells·mL–1. A complementary on-chip fluorescence readout (via Nile Red staining) provides visual corroboration and specificity support but exhibits reduced analytical sensitivity, approximately 4 orders of magnitude lower than the electrochemical approach, consistent with nanoparticle-induced quenching at lower cell counts. Across model and drinking-water matrices tested, the comparative performance trend is preserved, and the complete workflow can be completed in about 2 h from capture to readout. Taken together, these results present a comprehensive, internally consistent comparison on a single capture construct and clarify when electrochemistry versus fluorescence is fit-for-purpose in the context of immunomagnetic separation. The platform supports low-consumable testing with a reusable, unmodified electrochemical chip, is amenable to multiplexed array formats, and is readily compatible with portable potentiostats for on-site measurements.