Ultrasensitive Cascade Enzyme Immunoassay Electrochemical Microfluidic Chip for Early Screening of Prostate Cancer

Immunosensors offer great promise for cancer diagnostics owing to their high sensitivity, specificity, and potential for miniaturization. However, conventional electrochemical immunosensors often rely on single-step enzyme reactions and single-biomarker detection, limiting their signal amplification capacity and diagnostic accuracy for complex diseases such as prostate cancer. Here, we presented an ultrasensitive cascade enzyme immunoassay electrochemical microfluidic chip (CEE-CHIP) for the simultaneous detection of multiple prostate cancer biomarkers. The system employed primary antibody-functionalized magnetic metal-organic frameworks (Fe₃O₄@MOF), followed by target antigens binding specifically and glucose oxidase-labeled secondary antibodies conjugating to form stable sandwich immunocomplexes. The functionalized Fe₃O₄@MOF were magnetically positioned within a microfluidic channel and a subsequent cascade reaction involving glucose oxidase generates H₂O₂. H₂O₂ was detected by a Co₃O₄/MXene-modified working electrode, resulting in amplified electrochemical signals. This system allowed for the simultaneous detection of multiple tumor markers, including prostate-specific antigen and Engrailed 2, with detection limits as low as 0.154 and 0.146 pg/mL, respectively. With a broad linear range (0.001-100 ng/mL), excellent sensitivity, and minimal sample consumption, the CEE-CHIP represented a promising non-invasive, point-of-care diagnostic platform for early prostate cancer detection and broader clinical applications.