Chib Rahul, Mummert Mark, Bora Ilkay, Laursen Bo W, Shah Sunil, Pendry Robert, Gryczynski Ignacy, Borejdo Julian, Gryczynski Zygmunt, Fudala Rafal
Department of Cell Biology, Immunology and Microbiology, Center for Fluorescence Technologies and Nanomedicine, University of North Texas Health Science Center, Fort Worth, TX, 76107, USA.
Mental Sciences Institute, University of North Texas Health Science Center, Fort Worth, TX, 76107, USA.
Anal Bioanal Chem. 2016 May;408(14):3811-21. doi: 10.1007/s00216-016-9472-5. Epub 2016 Mar 18.
In this report, we have designed a rapid and sensitive, intensity-based ratiometric sensing as well as lifetime-based sensing probe for the detection of hyaluronidase activity. Hyaluronidase expression is known to be upregulated in various pathological conditions. We have developed a fluorescent probe by heavy labeling of hyaluronic acid with a new orange/red-emitting organic azadioxatriangulenium (ADOTA) fluorophore, which exhibits a long fluorescence lifetime (∼20 ns). The ADOTA fluorophore in water has a peak fluorescence lifetime of ∼20 ns and emission spectra centered at 560 nm. The heavily ADOTA-labeled hyaluronic acid (HA-ADOTA) shows a red shift in the peak emission wavelength (605 nm), a weak fluorescence signal, and a shorter fluorescence lifetime (∼4 ns) due to efficient self-quenching and formation of aggregates. In the presence of hyaluronidase, the brightness and fluorescence lifetime of the sample increase with a blue shift in the peak emission to its original wavelength at 560 nm. The ratio of the fluorescence intensity of the HA-ADOTA probe at 560 and 605 nm can be used as the sensing method for the detection of hyaluronidase. The cleavage of the hyaluronic acid macromolecule reduces the energy migration between ADOTA molecules, as well as the degree of self-quenching and aggregation. This probe can be efficiently used for both intensity-based ratiometric sensing as well as fluorescence lifetime-based sensing of hyaluronidase. The proposed method makes it a rapid and sensitive assay, useful for analyzing levels of hyaluronidase in relevant clinical samples like urine or plasma. Graphical Abstract Scheme showing cleavage of HA-ADOTA probe by hyaluronidase and the change in the emission spectrum of HA-ADOTA probe before and after cleavage by hyaluronidase.
在本报告中，我们设计了一种快速灵敏的基于强度的比率传感以及基于寿命的传感探针，用于检测透明质酸酶活性。已知透明质酸酶表达在各种病理状况下会上调。我们通过用一种新的橙色/红色发射有机氮杂二氧杂三角烯鎓（ADOTA）荧光团对透明质酸进行重标记，开发了一种荧光探针，该荧光团具有较长的荧光寿命（约20纳秒）。水中的ADOTA荧光团的峰值荧光寿命约为20纳秒，发射光谱以560纳米为中心。由于有效的自猝灭和聚集体形成，高度ADOTA标记的透明质酸（HA - ADOTA）在峰值发射波长（605纳米）处出现红移，荧光信号较弱且荧光寿命较短（约4纳秒）。在存在透明质酸酶的情况下，样品的亮度和荧光寿命增加，同时峰值发射蓝移至其原始波长560纳米。HA - ADOTA探针在560和605纳米处的荧光强度之比可作为检测透明质酸酶的传感方法。透明质酸大分子的裂解减少了ADOTA分子之间的能量迁移，以及自猝灭和聚集程度。该探针可有效地用于基于强度的比率传感以及基于荧光寿命的透明质酸酶传感。所提出的方法使其成为一种快速灵敏的测定方法，可用于分析尿液或血浆等相关临床样品中透明质酸酶的水平。图形摘要：显示透明质酸酶对HA - ADOTA探针的裂解以及透明质酸酶裂解前后HA - ADOTA探针发射光谱变化的示意图。
