Facile Antibody Immobilization on a Redox-Active Thionine-Functionalized Carbon Nanofiber Surface for Rapid Electrochemical Immunosensing of a Bioengineered Malaria Protein Biomarker

Antibodies that target the <i>Plasmodium falciparum</i> histidine-rich protein-II biomarker (<i>Pf</i>HRP-II) are being used in rapid diagnostic tests (RDTs) of malaria. <i>Pf</i>HRP-II levels associated with severe malaria are typically greater than 100 ng mL^-1. Unfortunately, genetic variations within the <i>Pf</i>HRP-II gene can reduce the reliability of these RDTs by affecting both sensitivity and specificity. In this study, we developed in-house antibodies against conserved C-terminal 105 amino acids of the <i>Pf</i>HRP-II biomarker to enhance malaria diagnosis using an electrochemical immunosensor technique. Unlike conventional electrochemical immunosensor assays, which use solution-phase enzyme-transducer systems like ferricyanide that suffer from poor current sensitivity and false positives, we constructed a heterogeneous electrochemical immunosensor. This sensor employs highly redox-active thionine (Th) immobilized on a carbon nanofiber (CNF)-based chemically modified electrode (CME) platform. The prepared CME was characterized using several physicochemical techniques, revealing that the oxygen-rich functional groups of CNF serve as active sites for effective antibody binding and immunosensing. Sequential modifications were performed using 2 μL volumes of the polyclonal antibody, antigen (<i>Pf</i>HRP-II), bioengineered monoclonal antibody, and horseradish peroxidase-coupled secondary antibody (Ab2HRP), with each step requiring an incubation time of 3-5 min, resulting in a total working time of 30 ± 5 min. The immunosensor demonstrated excellent sensing signals within a range of 250 pg/mL to 100 ng/mL <i>Pf</i>HRP-II, with a high current sensitivity of 0.813 μA/ng mL^-1. Control experiments with healthy rabbit and human blood serum samples showed no current response, ruling out false positive signals from the assay. For real-time application, high-performance electrochemical immunosensing of rabbit and human blood serum samples spiked with <i>Pf</i>HRP-II was demonstrated with high accuracy.