Nanozyme-Amplified Closed Bipolar Electrochemical Colorimetric Platform for Multiplexed Visual Detection of Sepsis Biomarkers

Abstract

Early diagnosis of sepsis urgently requires the development of novel electrochemical sensing platforms for rapid, sensitive, and multiplexed measurement of inflammatory biomarkers at the point of care. Herein, we report a closed bipolar electrochemical (cBPE) colorimetric platform that couples nanozyme-catalyzed redox cycling at the sensing pole with Prussian blue (PB) electrodeposition at the signal pole. Bimetallic Au@Pt nanozymes (Au core, porous Pt shell) catalyze the reduction of [Fe(CN)6]3– by ammonia borane (NH3·BH3) with a specific activity of 310 U mg–1, which is 12-fold higher than that of the Au core, and an apparent catalytic efficiency (kcat/Km) of 2.7 ×107 M–1 s–1, determined under the first-order kinetic regime. Sepsis biomarkers captured on immunomagnetic beads and labeled with detection antibody-conjugated Au@Pt nanozymes were magnetically enriched at the cBPE anode, where the nanozyme-generated [Fe(CN)6]4– was re-oxidized to [Fe(CN)6]3–, sustaining a redox cycle that amplifies the Faradaic current. Charge conservation across the cBPE converts this current into an equivalent cathodic flux in a physically separated signal chamber, where Fe3+ reduction drives deposition of a blue PB film whose grayscale intensity increases monotonically with target concentration and is read by the naked eye. The spatially addressable cBPE array allows interleukin-6 (IL-6), C-reactive protein (CRP), and serum amyloid A (SAA) to be assayed in parallel, with the limits of detection of 0.83, 2.69, and 1.82 ng mL–1, respectively. In clinical serum samples from healthy controls (n = 11) and sepsis patients (n = 11), all three biomarkers were significantly elevated in the sepsis patients, with an area under the ROC curve of 0.82–1.00, demonstrating discriminative performance in real samples. The proposed cBPE-based electrochemical colorimetric platform integrates catalytic signal amplification, wireless multiplexing, and visual readout, offering a practical route toward point-of-care detection of sepsis-associated biomarkers.