Nanophotonic biosensors offer exceptional sensitivity in the presence of strong background signals by enhancing and confining light in subwavelength volumes. In the field of nanophotonic biosensors, antenna-in-box (AiB) designs consisting of a nanoantenna within a nanoaperture have demonstrated remarkable single-molecule fluorescence detection sensitivities under physiologically relevant conditions. However, their full potential has not yet been exploited as current designs prohibit insightful correlative multicolor single-molecule studies and are limited in terms of throughput. Here, we overcome these constraints by introducing aluminum-based hexagonal close-packed AiB (HCP-AiB) arrays. Our approach enables the parallel readout of over 1000 HCP-AiBs with multicolor single-molecule sensitivity up to micromolar concentrations using an alternating three-color excitation scheme and epi-fluorescence detection. Notably, the high-density HCP-AiB arrays not only enable high-throughput studies at micromolar concentrations but also offer high single-molecule detection probabilities in the nanomolar range. We demonstrate that robust and alignment-free correlative multicolor studies are possible using optical fiducial markers even when imaging in the low millisecond range. These advancements pave the way for the use of HCP-AiB arrays as biosensor architectures for high-throughput multicolor studies on single-molecule dynamics.

中文翻译：

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用于高通量多色单分子研究的六角等离子体阵列


纳米光子生物传感器通过增强和限制亚波长体积内的光，在存在强背景信号的情况下提供卓越的灵敏度。在纳米光子生物传感器领域，由纳米孔径内的纳米天线组成的盒内天线（AiB）设计已在生理相关条件下表现出卓越的单分子荧光检测灵敏度。然而，它们的全部潜力尚未得到充分利用，因为当前的设计禁止深入的相关多色单分子研究，并且吞吐量受到限制。在这里，我们通过引入铝基六方密堆积 AiB (HCP-AiB) 阵列来克服这些限制。我们的方法使用交替三色激发方案和落射荧光检测，能够并行读出 1000 多个 HCP-AiB，其多色单分子灵敏度高达微摩尔浓度。值得注意的是，高密度 HCP-AiB 阵列不仅能够实现微摩尔浓度的高通量研究，而且还提供纳摩尔范围内的高单分子检测概率。我们证明，即使在低毫秒范围内成像，也可以使用光学基准标记进行稳健且无需对准的相关多色研究。这些进步为使用 HCP-AiB 阵列作为生物传感器架构进行单分子动力学高通量多色研究铺平了道路。