Innovative Aptamer-HRP Conjugation for Cardiac Troponin I Detection: A Novel ELASA Approach for AMI Diagnostics.

Abstract

Acute myocardial infarction (AMI), a major global cause of mortality, is diagnosed using cardiac troponin I (cTnI). Antibody-based assays face challenges, prompting the exploration of aptamers. This study develops an aptamer-HRP probe and ELASA for improved cTnI detection. Aptamer-based enzyme-linked aptamer assays (ELASA) were developed to detect cTnI using Tro4 and Tro6 aptamers. Molecular docking was performed via the HDOCK web server to confirm aptamer binding affinity to cTnI. Tro4 was biotinylated for use as a capture probe, while Tro6 was conjugated to HRP through sulfo-SMCC crosslinking, followed by size exclusion chromatography and purification. Direct and sandwich ELASA assays were performed using streptavidin-coated plates and clinical serum samples from AMI and non-AMI patients. Data were analyzed using GraphPad Prism10 and SPSS software. Molecular docking confirmed the high binding affinity of Tro4 and Tro6 aptamers to cTnI, with significant interaction energies. Direct ELASA verified aptamer binding, and optimal concentrations were determined as 10μM for Tro4 and 5μM for Tro6. A sandwich ELASA using paired aptamers achieved improved sensitivity and specificity for cTnI detection. The assay displayed a linear response between 0.1-22 ng.mL cTnI (R²=0.94), with a limit of detection (LOD) of 0.10ng.mL. When tested on patient serum samples, results correlated with a commercial antibody-based ELASA kit. This study successfully developed a highly sensitive and specific sandwich ELASA for cTnI detection, utilizing the optimal aptamers Tro4 and Tro6. The results demonstrated excellent sensitivity, specificity, and potential clinical applicability, offering a promising alternative to antibody-based assays.