en Biomarkers (diagnosis & treatment) Biomarkers (diagnosis & treatment) Original Article Original Article <p><span style="font-size:12px;"><span style="font-family:Tahoma;"><span style="line-height:14.0pt">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.</span><br> <span style="line-height:14.0pt">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. </span><br> <span style="line-height:14.0pt">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 10&mu;M for Tro4 and 5&mu;M 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&ndash;22 ng.mL cTnI (R&sup2;=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.</span><br> <span style="line-height:14.0pt">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.</span></span></span></p> Cardiac Troponin I (cTnI), Aptamer-based ELASA, Acute myocardial infarction (AMI), Biomarker detection 705 713 http://ijmcmed.org/browse.php?a_code=A-10-2635-3&slc_lang=en&sid=1 Mahshid Bahari ma.bahari@sbmu.ac.ir 100319475328460034473 100319475328460034473 No Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences Farshid Yeganeh fyeganeh@gmail.com 100319475328460034474 100319475328460034474 Yes Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences Ali Kargar Kheirabad kargarapgv@gmail.com 100319475328460034475 100319475328460034475 No Research and Development of Arghavan Teb Kavian Co