Wang Qin, Li Yanli, Yao Lige, Li Huiqin, Zhang Liuyan, Wang Yingjie, Li Jiayin, Chen Tian, Chai Kun, Gao Junli, Gao Junshun, Su Li, Li Xueming
The Third People's Hospital of Zhengzhou, Zhengzhou, 450000, China.
The First People's Hospital of Shangqiu, Shangqiu, 476000, China.
Anal Sci. 2025 Feb;41(2):119-126. doi: 10.1007/s44211-024-00673-w. Epub 2025 Jan 1.
TIMP-1 (Tissue Inhibitor of Metalloproteinases-1) is a protein involved in regulating extracellular matrix (ECM) degradation. It is recognized as a significant biomarker for cancer diagnosis. This study aimed to develop and characterize a single-stranded DNA (ssDNA) aptamer targeting human TIMP-1 protein with high affinity and specificity. A magnetic beads-based SELEX process combined with qPCR was used to select aptamers over seven rounds. The enriched ssDNA library was analyzed using high-throughput sequencing to identify candidate sequences, and these sequences were characterized using surface plasmon resonance (SPR) and binding assays to evaluate their affinity and specificity. The selected ssDNA aptamer demonstrated a dissociation equilibrium constant (K) of 0.41 nM and a very slow off-rate, enabling effective capture of TIMP-1 in serum samples. Furthermore, a chemiluminescent aptasensor was developed for TIMP-1 detection, which exhibited high specificity and a broad linear detection range from 1 to 500 ng/mL in human serum. The developed ssDNA aptamer targeting TIMP-1 shows high affinity and specificity, and the chemiluminescent aptasensor demonstrates promising potential for clinical diagnosis of TIMP-1 levels in human serum.
基质金属蛋白酶组织抑制因子-1（TIMP-1）是一种参与调节细胞外基质（ECM）降解的蛋白质。它被认为是癌症诊断的重要生物标志物。本研究旨在开发并表征一种对人TIMP-1蛋白具有高亲和力和特异性的单链DNA（ssDNA）适配体。基于磁珠的SELEX过程结合定量PCR用于经过七轮筛选适配体。使用高通量测序分析富集的ssDNA文库以鉴定候选序列，并使用表面等离子体共振（SPR）和结合试验对这些序列进行表征，以评估它们的亲和力和特异性。所选的ssDNA适配体表现出0.41 nM的解离平衡常数（K）和非常慢的解离速率，能够有效捕获血清样品中的TIMP-1。此外，还开发了一种用于TIMP-1检测的化学发光适配体传感器，该传感器在人血清中表现出高特异性和1至500 ng/mL的宽线性检测范围。所开发的靶向TIMP-1的ssDNA适配体显示出高亲和力和特异性，并且化学发光适配体传感器在临床诊断人血清中TIMP-1水平方面显示出有前景的潜力。
