Ultrasensitive\nElectrochemical Vertical Flow Immunoassay\nfor Rapid and Simultaneous Detection of Interleukin‑6 and Procalcitonin

Rapid and multiplexed detection of biomarkers plays an\nindispensable\nrole in disease diagnosis. Although paper-based lateral flow immunoassays\nhave been widely used in this field, the speediness and throughput\nare still challenging issues. Herein, an electrochemical vertical\nflow immunoassay device (eVFID) is fabricated for rapid, ultrasensitive,\nand multiplexed detection of inflammatory biomarkers. Working electrodes\nwith excellent electrochemical performance and permeability properties\nwere directly fabricated on the nitrocellulose membrane to enable\nboth the vertical flow of the sample solution and electrochemical\ndetection. This vertical configuration can remarkably improve the\nspeediness of the immunoassay and effectively inhibit the cross-talk\nreactions among immunomolecules, thus allowing rapid and simultaneous\ndetection of multiplexed biomarkers. Furthermore, a signal amplification\nstrategy based on horseradish peroxidase and tetramethylbenzidine\nwas integrated into the eVFID to substantially increase the sensitivity\nof the electrochemical detection. A low limit of detection of 0.1\nand 0.22 pg mL<sup>–1</sup> was obtained for two low-abundance\ninflammatory biomarkers, interleukin-6 (IL-6) and procalcitonin (PCT),\nrespectively. Finally, using a two-channel eVFID, simultaneous detection\nof IL-6 and PCT in human plasma samples was successfully realized\nwithin 5 min. We believe that the eVFID holds great promise for speedy\nand high-throughput biomarker detection at the point of care.