Microfluidic paper-based multiplex colorimetric immunodevice based on the catalytic effect of Pd/Fe3O4@C peroxidase mimetics on multiple chromogenic reactions

In this report, a non-toxic method was proposed for the simple synthesis of palladium nanoparticles (Pd)/Fe₃O₄@C peroxidase mimetics by virtue of in situ growth of Pd nanoparticles on Fe₃O₄@C magnetic nanoparticles. And a microfluidic paper-based multiplex colorimetric immunodevice (named α-sheet) was developed by site-selectively immobilizing multiple antigens owing to its intrinsic high-efficiency catalytic activity of peroxidase mimetics to multiple chromogenic reactions. The immunosensor platform was prepared by growing a layer of flower-like gold nanoparticles which could entrap the primary antibodies onto paper sensing zones, and the as-prepared Pd/Fe₃O₄@C peroxidase mimetics was used to label secondary antibodies. In the presence of 3,3′,5,5′-tetramethylbenzidine and o-phenylenediamine chromogenic substrates, Pd/Fe₃O₄@C peroxidase mimetics catalyzed chromogenic reactions and showed different colors with respective intensity. To precisely identify the intensity, a piece of black wax printed chromatographic paper with three observing windows (named β-sheet) was flatted on α-sheet. Under the optimal condition, the proposed multiplex colorimetric immunodevice displayed wide linear ranges from 0.005 to 30 ng mL⁻¹ with low detection limits of 1.7 pg mL⁻¹ for carcinoembryonic antigen (CEA) and α-fetoprotein (α-AFP). Meanwhile, the proposed method provided provided a non-toxic, low-cost and promising tool for point-of-care diagnosis.