Rapid quantitative analysis using a single molecule counting approach
Rapid quantitative analysis using a single molecule counting approach
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DOI：
10.1016/j.ab.2006.01.031
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发表时间：
2006-05-01
影响因子：
2.9
通讯作者：
Nalefski, EA
中科院分区：
文献类型：
--
作者：
D'Antoni, CM;Fuchs, M;Nalefski, EA
单分子检测由成对的荧光标记探针特异性结合的目标分子在生物分子的灵敏定量方面显示出巨大的潜力。到目前为止，还没有报告严格评估采用这种双探针方法的单分子检测平台的分析能力或其数据分析方法的性能。本文描述了一种快速、自动化、灵敏的多色单分子检测装置和一种基于符合事件计数的荧光探针油液检测的新扩展。该方法通过对同时随机穿过询问区的未结合探针进行校正，根据观察到的符合荧光事件的总数来估计感兴趣的双标记分子的数量。事件计数在三种不同荧光通道的组合上进行了评估，并被证明在对目标分子产生更广泛的线性动态响应方面优于传统的空间互相关。此外，该方法成功地检测了在复杂的RNA背景下与荧光标记的寡核苷酸探针杂交的模型RNA靶标的亚皮摩尔浓度。这些结果表明，所描述的荧光事件计数方法是快速、灵敏地定量分析任何样品分析物的通用工具，包括核酸和蛋白质，可以为其开发一对特定的探针。(C)2006 Elsevier Inc.保留所有权利。
Single molecule detection of target molecules specifically bound by paired fluorescently labeled probes has shown great potential for sensitive quantitation of biomolecules. To date, no reports have rigorously evaluated the analytical capabilities of a single molecule detection platform employing this dual-probe approach or the performance of its data analysis methodology. I it this paper, we describe a rapid, automated, and sensitive multicolor single molecule detection apparatus and a novel extension of coincident event counting based oil detection of fluorescent probes. The approach estimates the number of dual-labeled molecules of interest from the total number of coincident fluorescent events observed by correcting for unbound probes that randomly pass through the interrogation zone simultaneously. Event counting was evaluated on three combinations of distinct fluorescence channels and was demonstrated to Outperform conventional spatial cross-correlation in generating a wider linear dynamic response to target molecules. Furthermore, this approach Succeeded in detecting sub-picomolar concentrations of a model RNA target to which fluorescently labeled oligonucleotide probes were hybridized in a complex background of RNA. These results illustrate that the fluorescent event counting approach described represents a general tool for rapid sensitive quantitative analysis of any sample analyte, including nucleic acids and proteins, for which pairs of specific probes can be developed. (c) 2006 Elsevier Inc. All rights reserved.