【JACS】突破1000 nm！通过结构工程改造花菁染料以获得短波红外发射的J-聚集体#

Figure 1. Overview of benzothiazole J-aggregates reported herein. a) Cartoon representing the self-assembly of dye monomers upon J-aggregation, and previously reported characterization (density of states and TEM) of the dye Cy7-DPA. Reproduced from ref (32). Copyright 2020 American Chemical Society. b) Summary of the dyes and aggregates investigated in this work, including the effect of steric bulk, hypothesized packing arrangements, photophysical properties and applications in multiplexed imaging.#

Figure 2. Dye synthesis and photophysical characterization of solubilized dyes. a) Synthesis of sterically hindered cyanines 1a–d. b) Absorption spectra for 1a–d monomers in ethanol. c) Tabulated monomer dye photophysical properties in ethanol.#

通过控制染料浓度、溶剂比例、配制温度和组装动力学，研究人员从这四种亲缘分子中，惊人地隔离并表征了多达13种不同的近红外/短波红外聚集体，这一发现彻底打破了通常单一菁染料仅能形成一到两种聚集态的传统认知。在图3中，作者展示了系统性的动力学筛选过程，并将这些聚集体归纳命名为J1、J2、J3和J4四类主要介相。

Figure 3. J-aggregate mesophases from dyes 1a–1d. a) Absorption of 0.1 mM 1b in different MeOH/H2O composition solutions after 24 h. b) Kinetic absorption screening of 0.1 mM 1b in 5% MeOH/H2O 1b after different time points. c) Isolated aggregate absorption spectra for J1, J2, J3, and J4 of 1a–d.#

为了阐明这些复杂介相在纳米尺度上的结构差异，研究人员联合利用低温透射电子显微镜（Cryo-EM）和原子力显微镜（AFM）进行了深度剖析。如图4所示，Cryo-EM和AFM的高分辨率图像直观地揭示了四种聚集体介相的形貌特征。其中，J1、J3和J4介相均展现出平整的二维片状结构，但它们的单分子层厚度存在细微差异。最引人瞩目的是染料1b在特定溶剂条件下形成的J2聚集体，它呈现出均匀的、管径仅为5纳米的微米级单壁纳米管形貌，这也是科学界首次表征并证实可以在短波红外波段发光的管状J-聚集体。 利用线性二色性光谱和电磁激子哈密顿量模型，研究人员精确计算出该纳米管中单体偶极矩与圆柱轴线之间的手性夹角为43.5度。

Figure 4. Structural characterization of the mesophases of 1b. a) Cryo-EM images of J1–J4 aggregates of dye 1b, showing sheetlike morphologies for J1, J3, and J4, and a tubelike structure for J2. Scale bar: 200 nm. b) AFM images of J1–J4 aggregates of dye 1b, with black lines indicating where height profiles were measured, and color bars presenting height in nm. Scale bar: 200 nm. c) Fitting of height profiles extracted from the 1b J1 AFM image. d) AFM heights measured for each aggregate isolated at room temperature. e) Average size determined by 5–10 DLS measurements of each aggregate isolated at room temperature. For d and e, the red line indicates the median value, the box corresponds to the 25%–75% percentiles, the whiskers represent the smallest and largest values, and the red crosses are outliers. See Table S2 for tabulated structural data.#

这些聚集体介相之间并非孤立，而是存在着极其微妙的动态热力学与动力学平衡。如图5所示，动力学和热力学转化实验表明，J1是一种亚稳态的动力学捕获产物，随着时间的推移，J1会自发克服能垒转化为更加稳定但高度有序度稍降、展现出面外跃迁特征的J4热力学产物。而J2纳米管与二维片状J3之间则可以随着甲醇含量的微调而发生快速、可逆的相互转化，这表明两者具有相似的晶胞堆积方式，但由于溶剂化效应的细微变动而在宏观上展现出截然不同的空间曲率。

Figure 5. Interconversion of J-aggregate mesophases and structural model. a) Absorption profiles of 1b aggregates under three different preparation conditions (% MeOH) after varying aggregation times. b) Potential energy diagram showing the interconversions between mesophases at low (red) and high (black) methanol content. c) Cartoon depicting self-assembly for aggregates of 1b.#

为了在理论层面上验证空间位阻调控滑移角的最初假设，研究人员利用密度泛函理论（DFT）对单体二聚体堆积进行了计算。如图6所示，随着单体4’位空间位阻由苯基增大到叔丁基，使能量最低的二聚体滑移距离从4.6 Å增大到5.2 Å，从而使得J1聚集体的亮态更加贴近激子带边缘。这不仅在实验上完美解释了J1聚集体红移随位阻增大而增加的现象，也直接揭示了通过空间阻碍策略设计窄发光、强发射短波红外聚集体的科学合理性。

Figure 6. a) Structure, model, and photophysical trends of 1a and 1d with optimized slip values between neighboring monomers. b) Relationship between slip and J-coupling responsible for dye redshift with monopole (solid line) and point dipole (dotted line) theories modeled. c) Energy diagram of the J1 to J4 transition, which occurs more rapidly as steric hindrance increases.#

Figure 7. a) Emission of the isolated aggregates for 1a–1d using the same conditions stated in Figure 3c. b) Quantum yields of each of the aggregate mesophases, determined using relative measurements with Chrom7 as a standard. c) Multiplex imaging of the monomer and three aggregates of 1b in isolated capillaries via differential excitation. False-colored composite image followed by single-channel images. The single channels are as follows: 786 nm ex. (monomer, 0.05 mM in MeOH), 890 nm ex. (J2, 0.5 mM in 20% MeOH), 974 nm ex. (J3, 0.18 mM in 30% MeOH), 1060 nm ex. (J1, 0.083 mM in 5% MeOH). Images were taken with an InGaAs camera using 1200 nm LP filters and a 5.0 mm silicon window. d) Three-color image of the monomer and J-aggregates J1 and J2 of 1b within a mouse phantom. Individual single-color channel images and imaging parameters are shown in Figure S15. Scale bar = 10 mm.#

在生物成像应用方面，这13种新型聚集体中有9种在室温下具有短波红外发光能力。如图7所示，研究人员展示了这些聚集体的短波红外发射光谱与相对荧光量子产率。其中，染料1b在组装为J3二维片层时，量子产率达到了0.15%，这是目前同类短波红外菁染料聚集体中的最高值之一。利用染料1b单体及其三种不同的发射态，研究人员在不同波长激光的分别激发下，在毛细管和模拟生物组织的鼠标模型中实现了极低信号互扰的四色多通道成像。 这一成果证明了仅依靠单一化学小分子，通过自组装介相调控，即可实现等同于多种不同染料组合的多通道窄带宽成像效果。