Semiconductor nanowires can enhance the signal of fluorescent molecules, thus significantly improving the limits of fluorescence detection in optical biosensing. In this work, we explore how the sensitivity can further be enhanced through “digital” detection of adequately spaced vertically aligned nanowires, employing single-emitter localization methods, and bright-field microscopy. Additionally, we introduce a systematic analysis pipeline aimed at harnessing this digital detection capability and evaluate its impact on detection sensitivity. Using a streptavidin-biotin assay, we demonstrate that single-emitter localization expands the dynamic range to encompass five orders of magnitude, enabling detections of concentrations ranging from 10 fM to 10 nM. This represents two to three orders of magnitude improvement in detection compared to methods that do not utilize single-emitter localization. We validate our analysis framework by simulating an artificial dataset based on numerical solutions of Maxwell’s equations. Furthermore, we benchmark our results against total internal reflection fluorescence microscopy and find, in time-resolved titration experiments, that nanowires offer higher sensitivity at the lowest concentrations, attributed to a combination of higher protein capture rate and higher intensity per single protein binding event. These findings suggest promising applications of nanowires in both endpoint and time-resolved biosensing.

中文翻译：

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基于纳米线的分子光学检测的图像分析优化


半导体纳米线可以增强荧光分子的信号，从而显着提高光学生物传感中荧光检测的极限。在这项工作中，我们探索了如何通过采用单发射器定位方法和明场显微镜对足够间隔的垂直排列纳米线进行“数字”检测来进一步提高灵敏度。此外，我们引入了一个系统分析管道，旨在利用这种数字检测能力并评估其对检测灵敏度的影响。使用链霉亲和素-生物素测定，我们证明单发射器定位将动态范围扩展至五个数量级，从而能够检测 10 fM 至 10 nM 的浓度范围。与不利用单发射器定位的方法相比，这意味着检测性能提高了两到三个数量级。我们通过模拟基于麦克斯韦方程组数值解的人工数据集来验证我们的分析框架。此外，我们将我们的结果与全内反射荧光显微镜进行基准比较，发现在时间分辨滴定实验中，纳米线在最低浓度下提供更高的灵敏度，这归因于更高的蛋白质捕获率和每个单一蛋白质结合事件的更高强度的组合。这些发现表明纳米线在端点和时间分辨生物传感中具有广阔的应用前景。