Structure-guided development of an electrochemical aptasensor for Salmonella Typhi HlyE antigen detection using in silico and experimental approaches

Efficient and timely detection of biomarkers for typhoid fever is pivotal for improving treatment outcomes and preventing further transmission of the disease. This study addresses the pressing need for a rapid and accurate point-of-care test to detect Salmonella Typhi HlyE antigen, a vital biomarker for typhoid fever. Leveraging the growing prominence of computer-based simulations for aptamer-protein interactions modelling and electrochemical biosensors for their high sensitivity, this study presents the development of a novel electrochemical aptasensor designed to accurately and effectively detect Salmonella Typhi HlyE antigen in blood samples of typhoid patients. We previously identified and characterized selective aptamers against HlyE antigen, and identified AptHlyE97 as the aptamer with highest binding affinity and selectivity. In this study we showed that AptHlyE97 formed the most stable complex with HlyE in molecular docking and dynamics simulations and an unbound 5’ end to enable conjugation to the sensor surface. The aptasensor features AptHlyE97 conjugated via thiol immobilization to the surface of a screen-printed gold electrode, and utilizing potassium ferricyanide and potassium ferrocyanide for electrochemical detection. The aptasensor showed a significant (p < 0.001) square wave voltammetry (SWV) response to HlyE with a diagnostic performance of 100% sensitivity and 85.7% specificity against other bacterial communicable disease pathogens. Additionally, the aptasensor exhibited a strong linear response with a limit of detection (LoD) of 0.158 ng/mL. We present here a novel diagnostic aptasensor capable of efficiently and selectively detecting the HlyE antigen from Salmonella Typhi. This aptasensor provides a robust foundation for future research in typhoid diagnosis and also highlights the potential impact in advancing point-of-care detection strategies in community settings.