Direct Kinetic Fingerprinting for High-Accuracy Single-Molecule Counting of Diverse Disease Biomarkers.
Direct Kinetic Fingerprinting for High-Accuracy Single-Molecule Counting of Diverse Disease Biomarkers.
复制标题
DOI：
10.1021/acs.accounts.0c00621
复制
发表时间：
2021-01-19
影响因子：
18.3
通讯作者：
Walter NG
中科院分区：
文献类型：
--
作者：
Mandal S;Li Z;Chatterjee T;Khanna K;Montoya K;Dai L;Petersen C;Li L;Tewari M;Johnson-Buck A;Walter NG
检测和量化生物体液中的疾病生物标志物的方法具有高度的特异性和敏感性，在实现临床诊断(包括护理点测试)方面发挥着关键作用。最广泛使用的分子生物标记物包括蛋白质、核酸、激素、代谢物和其他小分子。虽然已经开发了许多分析生物标志物的方法，但由于分析性能不足、成本高和/或其他实际缺点，大多数技术在临床上的实施是具有挑战性的。例如，通过数字聚合酶链式反应和下一代测序(NGS)检测无细胞核酸(CfNA)生物标记物需要耗时的核酸提取步骤，经常引入酶扩增偏差，当需要高特异性时，成本可能会很高。虽然已经报道了几种检测cfNAs的无扩增方法，但这些技术通常存在特异性和敏感性低的问题。同时，蛋白质生物标记物的定量通常使用免疫分析方法，如酶联免疫吸附试验(ELISA)；这些方法的分析性能通常受到高亲和力和特异性的抗体的可用性以及抗体与分析表面的显著非特异性结合的限制。为了解决现有生物标志物检测方法的不足，建立一个能够检测不同类型分析物的通用诊断平台，我们开发了一种无扩增的方法，称为通过平衡泊松采样的单分子识别(SiMREPS)，用于检测具有任意高特异性和单分子灵敏度的各种生物标志物。SiMREPS利用荧光检测探针与固定的目标分子的瞬时、可逆结合来生成可由单分子荧光显微镜检测的动态指纹。对这些动力学指纹的分析可以近乎完美地区分与目标分子的特定结合和任何非特定结合。早期的概念验证研究表明，体外检测miRNAs的检测限(LOD)约为1fM，单核苷酸多态的选择性为>；500倍。随后，SiMREPS方法被扩展到从生物体液中检测罕见的突变等位基因，突变等位基因比例低至百万分之一，对应的特异度为99.99999%。最近，SiMREPS被推广到使用动态结合的抗体探针进行蛋白质定量，允许从低股分子到全分子范围内的LOD。最后，SiMREPS已被证明适用于培养细胞中miRNAs的原位检测，小分子毒素和药物的定量，以及单分子水平上的端粒酶活性监测。在本文中，我们讨论了SiMREPS用于分子分析物的高特异性和高灵敏度检测的原理，包括分析设计的考虑因素。我们讨论了SiMREPS用于检测非常不同的分析物的一般性，并提供了数据处理方法的概述，包括使用超分辨率分析扩展动态范围和使用深度学习算法改进性能。最后，我们描述了SiMREPS方法当前的挑战、机遇和未来方向。
Methods for detecting and quantifying disease biomarkers in biofluids with high specificity and sensitivity play a pivotal role in enabling clinical diagnostics, including point-of-care tests. The most widely used molecular biomarkers include proteins, nucleic acids, hormones, metabolites, and other small molecules. While numerous methods have been developed for analyzing biomarkers, most techniques are challenging to implement for clinical use due to insufficient analytical performance, high cost, and/or other practical shortcomings. For instance, the detection of cell-free nucleic acid (cfNA) biomarkers by digital PCR and next generation sequencing (NGS) requires time-consuming nucleic acid extraction steps, often introduces enzymatic amplification bias, and can be costly when high specificity is required. While several amplification-free methods for detecting cfNAs have been reported, these techniques generally suffer from low specificity and sensitivity. Meanwhile, the quantification of protein biomarkers is generally performed using immunoassays such as enzyme-linked immunosorbent assay (ELISA); the analytical performance of these methods is often limited by the availability of antibodies with high affinity and specificity, as well as the significant nonspecific binding of antibodies to assay surfaces. To address the drawbacks of existing biomarker detection methods and establish a universal diagnostics platform capable of detecting different types of analytes, we have developed an amplification-free approach, named single-molecule recognition through equilibrium Poisson sampling (SiMREPS), for the detection of diverse biomarkers with arbitrarily high specificity and single-molecule sensitivity. SiMREPS utilizes the transient, reversible binding of fluorescent detection probes to immobilized target molecules to generate kinetic fingerprints that are detected by single-molecule fluorescence microscopy. Analysis of these kinetic fingerprints enables near-perfect discrimination between specific binding to target molecules and any nonspecific binding. Early proof-of-concept studies demonstrated the in vitro detection of miRNAs with limits of detection (LOD) of approximately 1 fM and >500-fold selectivity for single-nucleotide polymorphisms. The SiMREPS approach was subsequently expanded to the detection of rare mutant DNA alleles from biofluids at mutant allele fractions as low as 1 in 1 million, corresponding to a specificity of >99.99999%. Recently, SiMREPS was generalized to protein quantification using dynamically binding antibody probes, permitting LODs in the low-femtomolar to attomolar range. Finally, SiMREPS has been demonstrated to be suitable for the in situ detection of miRNAs in cultured cells, the quantification of small-molecule toxins and drugs, and the monitoring of telomerase activity at the single-molecule level. In this Account, we discuss the principles of SiMREPS for the highly specific and sensitive detection of molecular analytes, including considerations for assay design. We discuss the generality of SiMREPS for the detection of very disparate analytes, and provide an overview of data processing methods, including the expansion of dynamic range using super-resolution analysis and the improvement of performance using deep learning algorithms. Finally, we describe current challenges, opportunities, and future directions for the SiMREPS approach.
登录
查看更多内容
影响因子：
8
作者：
Gazdar, A. F.
通讯作者：
Gazdar, A. F.
DOI：
10.1146/annurev-anchem-061516-045340
发表时间：
2017-01-01
期刊：
ANNUAL REVIEW OF ANALYTICAL CHEMISTRY, VOL 10
影响因子：
--
作者：
Cohen, Limor;Walt, David R.
通讯作者：
Walt, David R.
影响因子：
3.4
作者：
Floyd, Daniel L.;Harrison, Stephen C.;van Oijen, Antoine M.
通讯作者：
van Oijen, Antoine M.
影响因子：
9.9
作者：
Geyer PE;Holdt LM;Teupser D;Mann M
通讯作者：
Mann M
影响因子：
4
作者：
Chen, Guoli;Mosier, Stacy;Gocke, Christopher D.;Lin, Ming-Tseh;Eshleman, James R.
通讯作者：
Eshleman, James R.