Expanding the Dynamic Range of Fluorescence Assays through Single-Molecule Counting and Intensity Calibration.
Expanding the Dynamic Range of Fluorescence Assays through Single-Molecule Counting and Intensity Calibration.
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DOI：
10.1021/jacs.8b08879
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发表时间：
2018-10-24
影响因子：
15
通讯作者：
Smith AM
中科院分区：
文献类型：
--
作者：
Smith L;Kohli M;Smith AM
表面捕获分析可以测量1000倍浓度范围和亚纳摩尔水平的荧光标记分析物，但许多生物分子的丰度表现出1,000,000倍的变化，一直到毫微摩尔水平。这项工作的目标是通过使用成像来结合分子计数和单分子系综强度校准来扩大荧光分析的动态范围。我们评估表面捕获的荧光标记施加的光学限制，比较不同荧光团类别的性能，并使用探测器采集参数来跨越宽范围的荧光辐射。我们发现，荧光蛋白藻红蛋白提供了独特的合适的性质，具有异常强烈和均匀的单荧光团亮度，可以克服任意的斑点检测阈值偏差。然后，使用滚圆放大技术对与癌症相关的miRNA生物标记物进行密集标记，克服了非特定结合荧光团造成的主要限制，允许对近5个数量级的浓度进行准确的单分子检测和校准，检测下限为29 fM。这些成像和分子计数策略可以广泛应用于扩大各种表面荧光分析的检测极限和动态范围。
Surface capture assays can measure fluorescently labeled analytes across a 1,000-fold concentration range and at the sub-nanomolar level, but many biological molecules exhibit 1,000,000-fold variations in abundance down to the femtomolar level. The goal of this work is to expand the dynamic range of fluorescence assays by using imaging to combine molecular counting with single-molecule calibration of ensemble intensities. We evaluate optical limits imposed by surface-captured fluorescent labels, compare performances of different fluorophore classes, and use detector acquisition parameters to span wide ranges of fluorescence irradiance. We find that the fluorescent protein phycoerythrin provides uniquely suitable properties with exceptionally intense and homogeneous single-fluorophore brightness that can overcome arbitrary spot detection threshold biases. Major limitations imposed by nonspecifically bound fluorophores were then overcome using rolling circle amplification to densely label cancer-associated miRNA biomarkers, allowing accurate single-molecule detection and calibration across nearly 5 orders of magnitude of concentration with a detection limit of 29 fM. These imaging and molecular counting strategies can be widely applied to expand the limit of detection and dynamic range of a variety of surface fluorescence assays.
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影响因子：
48
作者：
Hua, Boyang;Han, Kyu Young;Zhou, Ruobo;Kim, Hajin;Shi, Xinghua;Abeysirigunawardena, Sanjaya C.;Jain, Ankur;Singh, Digvijay;Aggarwal, Vasudha;Woodson, Sarah A.;Ha, Taekjip
通讯作者：
Ha, Taekjip
影响因子：
16.6
作者：
Lim SJ;Zahid MU;Le P;Ma L;Entenberg D;Harney AS;Condeelis J;Smith AM
通讯作者：
Smith AM
影响因子：
2.2
作者：
Gadkar, Vijay J.;Filion, Martin
通讯作者：
Filion, Martin
影响因子：
7.4
作者：
Kairdolf, Brad A.;Mancini, Michael C.;Nie, Shuming
通讯作者：
Nie, Shuming
影响因子：
5.7
作者：
Frgala, Tomas;Kalous, Ondrej;Reynolds, C. Patrick
通讯作者：
Reynolds, C. Patrick