PDF(Pubmed)
Abstract：
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.
摘要：
纳米光子生物传感器通过增强和限制亚波长体积中的光，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中天线(AiB)设计已经证明在生理相关条件下具有显著的单分子荧光检测灵敏度。然而,它们的全部潜力尚未被开发，因为目前的设计禁止有洞察力的相关多色单分子研究，并且在通量方面受到限制。这里,我们通过引入铝基六边形密堆积AiB(HCP-AiB)阵列来克服这些限制。我们的方法使用交替的三色激发方案和落射荧光检测，可以并行读出超过1000个HCP-AiB，具有多达微摩尔浓度的多色单分子灵敏度。值得注意的是,高密度HCP-AiB阵列不仅能够在微摩尔浓度下进行高通量研究,而且在纳摩尔范围内提供高的单分子检测概率。我们证明，即使在低毫秒范围内成像，使用光学基准标记也可以进行稳健且无对准的相关多色研究。这些进展为使用HCP-AiB阵列作为生物传感器架构以单分子动力学的高通量多色研究铺平了道路。
