Assessing the stochastic intermittency of single quantum dot luminescence for robust quantification of biomolecules.
Assessing the stochastic intermittency of single quantum dot luminescence for robust quantification of biomolecules.
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DOI：
10.1021/ac4001332
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发表时间：
2013-07-16
影响因子：
7.4
通讯作者：
Walt, David R.
中科院分区：
文献类型：
--
作者：
Palacios, Manuel A.;Lacy, Michael M.;Schubert, Stephanie M.;Manesse, Mael;Walt, David R.
单分子检测方案承诺能够达到检测的极限：一个分子。在本文中，我们使用单半导体纳米晶体（量子点，QD）的随机发光来检测和定位粒子作为数字计数。这些数字计数可以与溶液中分析物的浓度相关。在这里，我们使用全内反射荧光（TIRF）显微镜探测固定在功能化基板上的单个量子点。量子点由于其显著的亮度和稳定性而在生物分析领域找到了自己的位置。尽管量子点具有许多突出的物理性质，但在单个粒子水平上显示出明显的间歇发光，这对单个粒子的检测提出了挑战。在本文中，我们证明了一种可靠的方法来检测量子点，利用这些信号波动的量子点的荧光信号的变化对背景信号的变化进行比较。这里开发的定量方法导致信号与背景的比率高达90：1，这是至少8倍，高于使用仅依赖于信号积分的方法获得的比率。这种增强的信号与背景比促进了稳健的阈值化过程，并导致检测限低于摩尔。
Single molecule detection schemes promise the ability to reach the ultimate limit of detection: one molecule. In this paper, we use the stochastic luminescence of single semiconductor nanocrystals (Quantum dots, QDs) to detect and localize particles as digital counts. These digital counts can be correlated to the concentration of analytes in solution. Here, we use Total Internal Reflection Fluorescence (TIRF) microscopy to probe individual QDs immobilized on a functionalized substrate. QDs have found their niche in the bioanalytical community due to their remarkable brightness and stability. Despite their numerous outstanding photophysical properties, QDs at the single particle level display a pronounced intermittent luminescence, posing a challenge for the detection of individual particles. In this paper we demonstrate a reliable method for detecting QDs that takes advantage of these signal fluctuations by comparing the variations in the QD’s fluorescence signals against variations of the background signal. The quantitative methodology developed here results in signal-to-background ratios up to 90:1, which is at least 8-times higher than the ratios obtained using methodologies relying solely on signal integration. This enhanced signal-to-background ratio facilitates a robust thresholding process and results infemtomolar limits of detection.
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