Innovative Aptamer-HRP Conjugation for Cardiac Troponin I Detection: ANovel ELASA Approach for AMI Diagnostics

Document Type : Original Article

Authors
Mahshid Bahari 1
Farshid Yeganeh 2
Ali Kargar 3
1 Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
2 Research and Development of Arghavan Teb Kavian Co, Tehran, Iran
3 Research and Development of Arghavan Teb Kavian Co, Tehran, Iran.
10.22088/IJMCM.BUMS.14.2.705
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.
Keywords: Cardiac Troponin I (cTnI), Aptamer-based ELASA, Acute myocardial
infarction (AMI), Biomarker detection
Keywords
Cardiac Troponin I (cTnI)
Aptamer-based ELASA
Acute myocardialinfarction (AMI)
Biomarker detection