Applied Research and Technology, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, Illinois 60064, United States of America.
Methods Appl Fluoresc. 2021 Mar 23;9(2):025006. doi: 10.1088/2050-6120/abeed8.
Acridinium 9-carboxylic acid derivatives have been extensively used as chemiluminescent labels in diagnostic assays. Triggering acridinium with basic hydrogen peroxide produces a highly strained dioxetanone intermediate, which converts into an acridone in an electronically excited state and emits light at 420-440 nm. Here, we introduce a novel acridinium-fluorescein construct emitting at 530 nm, in which fluorescein is covalently attached to the acridinium N-10 nitrogen via a propyl sulfonamide linker. To characterize the spectral properties of the acridinium-fluorescein chemiluminophores, we synthesized the analogous acridone-fluorescein constructs. Both acridinium and acridone were linked to either 5- or 6-carboxyfluorescein and independently synthesized as individual structural isomers. Using fluorescent acridone-fluorophore tandems, we investigated and optimized the diluent composition to prevent dye aggregation. As monomolecular species, the acridone isomers demonstrated similar absorption, excitation, and emission spectra, as well as the expected fluorescence lifetimes and molecular brightness. Chemical triggering of acridinium-fluorescein tandems, as well as direct excitation of their acridone-fluorescein analogs, resulted in a nearly complete energy transfer from acridone to fluorescein. Acridone-based dyes can be studied with steady-state spectroscopy. Thus, they will serve as useful tools for structure and solvent optimizations, as well as for studying chemiluminescent energy transfer mechanisms in related acridinium-fluorophore tandems. Direct investigations of the light-emitting molecules generated in the acridinium chemiluminescent reaction empower further development of chemiluminescent labels with red-shifted emission. As illustrated by the two-color HIV model immunoassay, such labels can find immediate applications for multicolor detection in clinical diagnostic assays.
吖啶 9-羧酸衍生物已广泛用作诊断分析中的化学发光标记物。用碱性过氧化氢引发吖啶会产生高度应变的二噁烷酮中间体，该中间体在电子激发态下转化为吖啶酮并发射 420-440nm 的光。在这里，我们引入了一种新型的吖啶-荧光素构建体，其发射波长为 530nm，其中荧光素通过丙基磺酰胺键连接到吖啶的 N-10 氮上。为了表征吖啶-荧光素化学发光体的光谱特性，我们合成了类似的吖啶酮-荧光素构建体。吖啶和吖啶酮分别与 5-或 6-羧基荧光素相连，并独立合成作为单个结构异构体。使用荧光吖啶酮-荧光团串联，我们研究并优化了稀释剂组成以防止染料聚集。作为单分子物种，吖啶异构体表现出相似的吸收、激发和发射光谱，以及预期的荧光寿命和分子亮度。吖啶-荧光素串联物的化学引发以及其吖啶酮-荧光素类似物的直接激发导致吖啶酮几乎完全向荧光素转移能量。吖啶基染料可以用稳态光谱进行研究。因此，它们将作为结构和溶剂优化以及研究相关吖啶-荧光团串联物中的化学发光能量转移机制的有用工具。直接研究吖啶化学发光反应中产生的发光分子可以进一步开发具有红移发射的化学发光标记物。如双色 HIV 模型免疫分析所示，此类标记物可立即应用于临床诊断分析中的多色检测。
