Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China.
Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China.
Biosens Bioelectron. 2023 Oct 15;238:115578. doi: 10.1016/j.bios.2023.115578. Epub 2023 Aug 6.
Quantification of microRNAs (miRNAs) at the single-molecule level is of great significance for clinical diagnostics and biomedical research. The challenges lie in the limits to transforming single-molecule measurements into quantitative signals. To address these limits, here, we report a new approach called a Single Microbead-based Space-confined Digital Quantification (SMSDQ) to measure individual miRNA molecules by counting gold nanoparticles (AuNPs) with localized surface plasmon resonance (LSPR) light-scattering imaging. One miRNA target hybridizes with the alkynyl-modified capture DNA probe immobilized on a microbead (60 μm) and the azide-modified report DNA probe anchored on AuNP (50 nm), respectively. Through the click reaction between the alkynyl and azide group, a single microbead can covalently link the AuNPs in the confined space within the view of the microscope. By digitally counting the light-scattering spots of AuNPs, we demonstrated the proposed approach with single-molecule detection sensitivity and high specificity of single-base discrimination. Taking the advantages of ultrahigh sensitivity, specificity, and the digital detection manner, the approach is suitable for evaluating cell heterogeneity and small variations of miRNA expression and has been successfully applied to direct quantification of miRNAs in one-tenth single-cell lysates and serum samples without RNA-isolated and nucleic acid amplification steps.
在单分子水平上对 microRNAs（miRNAs）进行定量分析对于临床诊断和生物医学研究具有重要意义。其挑战在于如何将单分子测量转化为定量信号。针对这些限制，我们提出了一种新的方法，称为基于单个微球的空间限制数字定量（SMSDQ），通过使用局部表面等离子体共振（LSPR）光散射成像来计数金纳米粒子（AuNPs）来测量单个 miRNA 分子。一个 miRNA 靶标分别与固定在微球（60μm）上的炔基修饰的捕获 DNA 探针和锚定在 AuNP（50nm）上的叠氮修饰的报告 DNA 探针杂交。通过炔基和叠氮基团之间的点击反应，单个微球可以在显微镜视野内的受限空间中将 AuNPs 共价连接起来。通过对 AuNP 的光散射斑点进行数字计数，我们展示了该方法具有单分子检测灵敏度和单碱基区分的高特异性。该方法具有超高灵敏度、特异性和数字检测方式的优势，适用于评估细胞异质性和 miRNA 表达的微小变化，并已成功应用于无需 RNA 分离和核酸扩增步骤的十分之一单细胞裂解物和血清样本中 miRNA 的直接定量。
