Wei Ai-Ping, Herron James N
Department of Pharmaceutics, University of Utah, Salt Lake City 84112, USA.
J Mol Recognit. 2002 Sep-Oct;15(5):311-20. doi: 10.1002/jmr.593.
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-(CH(2))(n)-T. These F-(CH(2))(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.
抗荧光素抗体对荧光的淬灭作用（高达98%）在文献中有充分记载。在此，我们报道一种系统，其中双荧光团分子经设计后，与抗荧光素抗体结合时荧光增强，而非淬灭。双荧光团分子由荧光素（F）通过不同数量的亚甲基单元与四甲基罗丹明（T）相连构成，记为F-(CH₂)ₙ-T。这些F-(CH₂)ₙ-T缀合物在溶液中游离时，由于分子内二聚化和堆积作用，几乎不发荧光。然而，与抗荧光素抗体结合后，罗丹明部分的荧光增强了110倍。这种增强被认为是由于分子内二聚体解离，解除了对罗丹明荧光的淬灭。另一方面，由于结合和分子内共振能量转移，荧光素的荧光仍保持淬灭状态。此外，激发波长处于荧光素的吸收最大值处，产生约90 nm的斯托克斯位移。该系统将分子识别与荧光发射的同时增强直接耦合，省去了大多数检测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。
