Department of Oncology , Mayo Clinic , Rochester , Minnesota 55905 , United States.
J Am Chem Soc. 2018 Oct 24;140(42):13904-13912. doi: 10.1021/jacs.8b08879. Epub 2018 Oct 12.
Surface capture assays can measure fluorescently labeled analytes across a 1000-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.
表面捕获分析可以测量荧光标记分析物在 1000 倍浓度范围内和亚纳摩尔水平下的浓度，但许多生物分子的丰度在皮摩尔水平下存在 100 万倍的变化。这项工作的目标是通过使用成像将分子计数与整体强度的单分子校准相结合，来扩展荧光分析的动态范围。我们评估了表面捕获荧光标记物所施加的光学限制，比较了不同荧光团类别的性能，并使用探测器采集参数来跨越广泛的荧光辐照度范围。我们发现，荧光蛋白藻红蛋白具有独特的适宜性质，具有异常强烈和均匀的单荧光团亮度，可以克服任意点检测阈值偏差。然后，使用滚环扩增来克服非特异性结合荧光团施加的主要限制，从而可以对与癌症相关的 miRNA 生物标志物进行密集标记，允许在近 5 个浓度数量级范围内进行准确的单分子检测和校准，检测限为 29 fM。这些成像和分子计数策略可以广泛应用于扩展各种表面荧光分析的检测限和动态范围。
