Bahari Mahshid, Yeganeh Farshid, Kargar Ali
Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Research and Development of Arghavan Teb Kavian Co, Tehran, Iran.
Int J Mol Cell Med. 2025 Jul 1;14(2):705-713. doi: 10.22088/IJMCM.BUMS.14.2.705. eCollection 2025.
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.
急性心肌梗死（AMI）是全球主要的死亡原因之一，通常使用心肌肌钙蛋白I（cTnI）进行诊断。基于抗体的检测方法面临挑战，这促使人们探索适体。本研究开发了一种适体-辣根过氧化物酶（HRP）探针和酶联适体吸附测定法（ELASA），以改进cTnI检测。开发了基于适体的酶联适体测定法（ELASA），使用Tro4和Tro6适体检测cTnI。通过HDOCK网络服务器进行分子对接，以确认适体与cTnI的结合亲和力。将Tro4生物素化用作捕获探针，而Tro6通过磺基琥珀酰亚胺基-4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯（sulfo-SMCC）交联与HRP偶联，然后进行尺寸排阻色谱和纯化。使用链霉亲和素包被的酶标板以及来自AMI和非AMI患者的临床血清样本进行直接和夹心ELASA测定。使用GraphPad Prism10和SPSS软件分析数据。分子对接证实了Tro4和Tro6适体与cTnI具有高结合亲和力，且相互作用能显著。直接ELASA验证了适体结合，并确定Tro4的最佳浓度为10μM，Tro6的最佳浓度为5μM。使用配对适体的夹心ELASA在cTnI检测中实现了更高的灵敏度和特异性。该测定法在0.1-22 ng/mL cTnI之间显示出线性响应（R²=0.94），检测限（LOD）为0.10 ng/mL。在患者血清样本上进行测试时，结果与基于商业抗体的ELASA试剂盒相关。本研究成功开发了一种用于cTnI检测的高灵敏度和特异性夹心ELASA，利用了最佳适体Tro4和Tro6。结果显示出优异的灵敏度、特异性和潜在的临床适用性，为基于抗体的检测方法提供了一种有前景的替代方案。
