Bifluorophoric molecules as fluorescent beacons for antibody-antigen binding.
Bifluorophoric molecules as fluorescent beacons for antibody-antigen binding.
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双荧光分子作为抗体-抗原结合的荧光信标。
DOI：
10.1002/jmr.593
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发表时间：
2002
期刊：
影响因子：
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通讯作者：
Herron,JamesN
中科院分区：
文献类型：
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作者：
Wei,Ai-Ping;Herron,JamesN
文献中充分记录了抗荧光素抗体对荧光的猝灭（高达98%）。在这里，我们报告了一个系统，其中，而不是淬灭，双荧光分子被设计成在与抗荧光素抗体结合时增加荧光。双荧光分子由荧光素（F）通过不同数量的亚甲基单元连接到四甲基罗丹明（T）制成，表示为F-（CH 2）n-T。这些F-（CH 2）n-T缀合物在溶液中游离时由于分子内二聚化和堆叠而几乎无荧光。然而，在与抗荧光素抗体结合后，从罗丹明部分观察到荧光增加高达110倍。这种增加被认为是由于分子内二聚体解离，使罗丹明荧光去猝灭。另一方面，黄绿素荧光由于结合和分子内共振能量转移而保持猝灭。此外，激发波长在荧光素的最大吸收处，得到约90 nm的斯托克位移。 该系统将分子识别与荧光发射的同时增加直接耦合，避免了大多数测定所需的洗涤和孵育步骤。它是开发均相检测的重要分子报告系统。版权所有© 2002年约翰威利父子有限公司。
The quenching of fluorescence (up to 98%) by anti‐fluorescein antibodies is well documented in the literature. Here we report a system where, instead of quenching, bifluorophoric molecules are designed to increase in fluorescence upon binding by an anti‐fluorescein antibody. Bifluorophoric molecules are made of fluorescein (F) linked to tetramethylrhodamine (T) via varying numbers of methylene units, denoted as F‐(CH2)n‐T. These F‐(CH2)n‐T conjugates are almost nonfluorescent when free in solution due to intramolecular dimerization and stacking. Upon binding to an anti‐fluorescein antibody, however, up to 110‐fold increase in fluorescence was observed from the rhodamine moiety. This increase is believed to result from intramolecular dimer dissociation that dequenches the rhodamine fluorescence. Fluorescein fluorescence, on the other hand, remains quenched due to binding and intramolecular resonance energy transfer. Moreover, the excitation wavelength was at the absorption maxima of fluorescein, giving a Stoke's shift of about 90 nm. This system couples directly molecular recognition with a concurrent increase in fluorescence emission, obviating wash and incubation steps required by most assays. It is an important molecular reporter system for developing homogeneous assays. Copyright © 2002 John Wiley & Sons, Ltd.