Sørensen Thomas Just, Thyrhaug Erling, Szabelski Mariusz, Luchowski Rafal, Gryczynski Ignacy, Gryczynski Zygmunt, Laursen Bo W
Department of Molecular Biology and Immunology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Fort Worth, TX 76107, USA. Nano-Science Center and Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 København Ø, Denmark.
Methods Appl Fluoresc. 2013 Apr 3;1(2):025001. doi: 10.1088/2050-6120/1/2/025001.
Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.
在众多可用的光学生物测定方法中，荧光各向异性传感具有很大优势，因为它提供了一种灵敏、仪器简单的比率检测方法。然而，它受到一个严重限制，即标记物的发射寿命需要与被标记生物分子的相关寿命（翻滚时间）相当。对于中等大小的蛋白质，这大约在20 - 200纳秒范围内，由于实际问题，目前限制了标记物的选择仅为丹磺酰类染料和某些芳香族染料。这些染料有显著缺点，即紫外/蓝光吸收和发射，以及通常较大的溶剂敏感性。在此，我们报道了一种基于氮杂二氧杂三角烯鎓基序的用于高分子量生物分子测定的新型荧光标记物的合成与表征。长荧光寿命、发红光的荧光团氮杂二氧杂三角烯鎓（ADOTA - NHS）的N - 羟基琥珀酰亚胺酯与抗兔免疫球蛋白G（抗IgG）偶联。利用长荧光寿命，通过稳态荧光各向异性和时间分辨方法测定高分子量抗体及其与兔免疫球蛋白G（IgG）复合物的相关时间：采用稳态或时间分辨荧光各向异性进行溶液相免疫测定。通过进行变温实验确定，配体的结合导致相关时间增加超过75%，稳态各向异性增加18%。结果表明，三角烯鎓类染料可用于各向异性测定，以检测涉及比大多数其他发红光有机染料所能检测的大得多的生物分子的结合事件。
