Four-color alternating-laser excitation single-molecule fluorescence spectroscopy for next-generation biodetection assays.
Four-color alternating-laser excitation single-molecule fluorescence spectroscopy for next-generation biodetection assays.
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DOI：
10.1373/clinchem.2011.176958
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发表时间：
2012-04
影响因子：
9.3
通讯作者：
Reitmair A
中科院分区：
文献类型：
--
作者：
Yim SW;Kim T;Laurence TA;Partono S;Kim D;Kim Y;Weiss S;Reitmair A
单分子检测 (SMD) 技术非常适合临床诊断应用，可以最大限度地减少宝贵的患者样本需求，同时最大限度地增加临床信息内容。然而，目前还没有一种基于 SMD 的通用平台技术，该技术允许对核酸和蛋白质靶标进行多重检测，并且适合自动化和集成到临床实验室工作流程中。我们使用了一种灵敏、特异、定量且经济高效的均质 SMD 方法，该方法具有高单井复用潜力，并使用交替激光激发 (ALEX) 荧光辅助分子分选扩展到 4 种颜色 (4c-ALEX)。识别分子用不同颜色的荧光染料标记，并且≥2种颜色的同时共焦检测构成阳性目标检测事件。实际上排除了大多数背景噪音源，从而消除了清洗步骤。利用多维探针化学计量 (S) 和单分子荧光共振能量转移效率 (E) 对分子进行分类，可以同时区分众多目标。我们在一个孔中展示了 (a) 25 种不同的荧光标记 DNA 的检测、区分和定量； (b) 8 种细菌遗传标记，包括在 11 种引起败血症的葡萄球菌和肠球菌菌株中发现的 3 种抗生素耐药性决定因素； (c) 血液中存在 6 种肿瘤标志物。结果表明，通过核酸和蛋白质的多重检测，可用于临床分子诊断应用，并表明其在癌症、传染病和其他疾病的早期诊断以及个性化医疗中的潜在用途。未来集成其他技术组件，以最大限度地减少分析前样品操作，同时最大限度地提高通量，应该能够为下一代医疗诊断测试开发用户友好的（“样品输入，答案输出”）护理点平台，从而大大节省成本和患者样品。
Single-molecule detection (SMD) technologies are well suited for clinical diagnostic applications by offering the prospect of minimizing precious patient sample requirements while maximizing clinical information content. Not yet available, however, is a universal SMD-based platform technology that permits multiplexed detection of both nucleic acid and protein targets and that is suitable for automation and integration into the clinical laboratory work flow. We have used a sensitive, specific, quantitative, and cost-effective homogeneous SMD method that has high single-well multiplexing potential and uses alternating-laser excitation (ALEX) fluorescenceaided molecule sorting extended to 4 colors (4c-ALEX). Recognition molecules are tagged with different-color fluorescence dyes, and coincident confocal detection of ≥2 colors constitutes a positive target-detection event. The virtual exclusion of the majority of sources of background noise eliminates washing steps. Sorting molecules with multidimensional probe stoichiometries (S) and single-molecule fluorescence resonance energy transfer efficiencies (E) allows differentiation of numerous targets simultaneously. We show detection, differentiation, and quantification—in a single well—of (a) 25 different fluorescently labeled DNAs; (b) 8 bacterial genetic markers, including 3 antibiotic drug–resistance determinants found in 11 septicemia-causing Staphylococcus and Enterococcus strains; and (c) 6 tumor markers present in blood. The results demonstrate assay utility for clinical molecular diagnostic applications by means of multiplexed detection of nucleic acids and proteins and suggest potential uses for early diagnosis of cancer and infectious and other diseases, as well as for personalized medicine. Future integration of additional technology components to minimize preanalytical sample manipulation while maximizing throughput should allow development of a user-friendly (“sample in, answer out”) point-of-care platform for next-generation medical diagnostic tests that offer considerable savings in costs and patient sample.
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影响因子：
15
作者：
Lee, Nam Ki;Koh, Hye Ran;Kim, Seong Keun
通讯作者：
Kim, Seong Keun
影响因子：
48
作者：
Campos, Luis A.;Liu, Jianwei;Munoz, Victor
通讯作者：
Munoz, Victor
DOI：
10.1073/pnas.0508584102
发表时间：
2005-11-29
影响因子：
11.1
作者：
Laurence, TA;Kong, XX;Weiss, S
通讯作者：
Weiss, S
通过磁珠和液滴蒸发的两步富集方法提高 DNA 检测灵敏度
影响因子：
2
作者：
Du, Xing-Li;Duan, De-Min;Li, Jiong
通讯作者：
Li, Jiong
影响因子：
4.8
作者：
Laurence, Ted A.;Kwon, Youngeun;Barsky, Daniel
通讯作者：
Barsky, Daniel