Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels
Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels
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DOI：
10.1038/s41565-024-01672-8
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发表时间：
2024-05-15
影响因子：
38.3
通讯作者：
Squires，Allison H.
中科院分区：
文献类型：
--
作者：
Chu，Jiachong;Ejaz，Ayesha;Squires，Allison H.
单分子水平的多重实时荧光检测可以揭示混合物和其他复杂样品中多个分子物种的化学计量、动力学和相互作用。然而，由于低信噪比、高光谱重叠和需要保持染料的化学相容性，基于荧光的传感通常限于一次仅检测3-4种颜色。在这里，我们设计了一个由几十个复合荧光标记组成的调色板，称为FRET荧光剂，用于单分子水平的多路光谱测量。FRET荧光是由三种化学成分（DNA，Cy 3和Cy 5）构成的紧凑纳米结构，由于几何形状，荧光团附着化学和DNA序列的变化，具有可调的光谱特性。我们证明了FRET荧光标记和检测低浓度（<100 fM）的mRNA，双链DNA和蛋白质的混合物使用反布朗电动陷阱。除了识别每个FRET fluor的独特光谱特征外，该陷阱还可以区分附着在目标上的FRET fluor和未结合的FRET fluor，从而实现免清洗传感。虽然通常被认为是一个不希望的并发症的荧光，在这里的固有灵敏度的荧光团的局部物理化学环境提供了一个新的设计轴互补改变FRET效率。因此，可区分的FRET荧光标记的数量可以组合地增加，同时保持化学相容性，使用最小的化学构建块集在单分子水平上扩展光谱复用的前景。
Multiplexed, real-time fluorescence detection at the single-molecule level can reveal the stoichiometry, dynamics and interactions of multiple molecular species in mixtures and other complex samples. However, fluorescence-based sensing is typically limited to the detection of just 3–4 colours at a time due to low signal-to-noise ratio, high spectral overlap and the need to maintain the chemical compatibility of dyes. Here we engineered a palette of several dozen composite fluorescent labels, called FRETfluors, for multiplexed spectroscopic measurements at the single-molecule level. FRETfluors are compact nanostructures constructed from three chemical components (DNA, Cy3 and Cy5) with tunable spectroscopic properties due to variations in geometry, fluorophore attachment chemistry and DNA sequence. We demonstrate FRETfluor labelling and detection for low-concentration (<100 fM) mixtures of mRNA, dsDNA and proteins using an anti-Brownian electrokinetic trap. In addition to identifying the unique spectroscopic signature of each FRETfluor, this trap differentiates FRETfluors attached to a target from unbound FRETfluors, enabling wash-free sensing. Although usually considered an undesirable complication of fluorescence, here the inherent sensitivity of fluorophores to the local physicochemical environment provides a new design axis complementary to changing the FRET efficiency. As a result, the number of distinguishable FRETfluor labels can be combinatorically increased while chemical compatibility is maintained, expanding prospects for spectroscopic multiplexing at the single-molecule level using a minimal set of chemical building blocks.