An electrochemical sensing platform based on targeted paired binding for biomarker detection in human serum samples.

Electrochemical analysis provides key technical foundations for the advancement of detection technologies, offering advantages such as device miniaturization, rapid responsiveness, cost-effectiveness, and capacity for real-time monitoring. A major challenge currently under investigation is that the interfacial reactions between the electrode surface and analytes hinder the discrimination of specific and nonspecific molecules. This study aimed to introduce a novel molecular detection platform using targeted paired binding and electrochemical sensing for the precise detection of zinc-alpha-2-glycoprotein 1 (AZGP1) in clinical samples. We introduced a biorecognition molecule (BRM) targeting AZGP1 to confer selectivity. An AZGP1-specific aptamer was isolated using the systematic evolution of ligands by the exponential enrichment technique. As a novel BRM, this aptamer demonstrated high sensitivity and specificity toward AZGP1, while also allowing facile chemical synthesis and low-cost modification. Nevertheless, when used for evaluating complex clinical samples, single-material BRMs often show limitations in anti-interference capacity and stability. Although developing antibodies requires immunization of animals and incurs higher storage and transportation costs, these antibodies remain the gold standard for biomolecular recognition in clinical testing applications. We further used a sandwich assay based on biolayer interferometry technology to identify monoclonal antibodies that paired with the aptamer for cooperative AZGP1 binding. This paired biorecognition molecular layer integrated the complementary strengths of antibodies and aptamers, including high sensitivity, programmable modifiability, molecular dynamics selectivity upon target interaction, and stable interfacial sensing signals in electrochemical analysis.