State Key Laboratory of Fine Chemicals, Department of Chemistry, Dalian University of Technology, Dalian 116012, China.
Anal Chem. 2010 Mar 15;82(6):2529-35. doi: 10.1021/ac100021m.
The time-resolved luminescence bioassay technique using luminescent lanthanide complexes as labels is a highly sensitive and widely used bioassay method for clinical diagnostics and biotechnology. A major drawback of the current technique is that the luminescent lanthanide labels require UV excitation (typically less than 360 nm), which can damage living biological systems and is holding back further development of time-resolved luminescence instruments. Herein we describe two approaches for preparing a visible-light-sensitized Eu(3+) complex in aqueous media for time-resolved fluorometric applications: a dissociation enhancement aqueous solution that can be excited by visible light for ethylenediaminetetraacetate (EDTA)-Eu(3+) detection and a visible-light-sensitized water-soluble Eu(3+) complex conjugated bovine serum albumin (BSA) for biolabeling and time-resolved luminescence bioimaging. In the first approach, a weakly acidic aqueous solution consisting of 4,4'-bis(1'',1'',1'',2'',2'',3'',3''-heptafluoro-4'',6''-hexanedion-6''-yl)-o-terphenyl (BHHT), 2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine (DPBT), and Triton X-100 was prepared. This solution shows a strong luminescence enhancement effect for EDTA-Eu(3+) with a wide excitation wavelength range from UV to visible light (a maximum at 387 nm) and a long luminescence lifetime (520 micros), to provide a novel dissociation enhancement solution for time-resolved luminescence detection of EDTA-Eu(3+). In the second approach, a ternary Eu(3+) complex, 4,4'-bis(1'',1'',1'',2'',2'',3'',3''-heptafluoro-4'',6''-hexanedion-6''-yl)-chlorosulfo-o-terphenyl (BHHCT)-Eu(3+)-DPBT, was covalently bound to BSA to form a water-soluble BSA-BHHCT-Eu(3+)-DPBT conjugate. This biocompatible conjugate is of the visible-light excitable feature in aqueous media with a wide excitation wavelength range from UV to visible light (a maximum at 387 nm), a long luminescence lifetime (460 micros), and a higher quantum yield (27%). The conjugate was successfully used for streptavidin (SA) labeling and time-resolved luminescence imaging detection of three environmental pathogens, Giardia lamblia , Cryptosporidium muris , and Cryptosporidium parvum , in water samples. Our strategy gives a general idea for designing a visible-light-sensitized Eu(3+) complex for time-resolved luminescence bioassay applications.
基于镧系元素配合物的时间分辨荧光生物分析技术是一种高灵敏度、广泛应用于临床诊断和生物技术的生物分析方法。目前该技术的主要缺点是镧系元素标记物需要紫外光激发（通常小于 360nm），这会对活的生物系统造成损害，阻碍了时间分辨荧光仪器的进一步发展。在此，我们描述了两种在水相介质中制备可见光敏化 Eu(3+)配合物用于时间分辨荧光应用的方法：一种是可以用可见光激发的用于乙二胺四乙酸（EDTA）-Eu(3+)检测的解离增强水溶液，另一种是用于生物标记和时间分辨荧光生物成像的水溶性可见光敏化 Eu(3+)配合物连接牛血清白蛋白（BSA）。在第一种方法中，制备了一种弱酸性水溶液，其中包含 4,4'-双（1'',1'',1'',2'',2'',3'',3''-七氟-4'',6''-己二酮-6''-基）-o-三联苯（BHHT）、2-（N,N-二乙基苯胺-4-基）-4,6-双（3,5-二甲基吡唑-1-基）-1,3,5-三嗪（DPBT）和 Triton X-100。该溶液对 EDTA-Eu(3+)具有很强的荧光增强效应，激发波长范围从紫外光到可见光（最大在 387nm），荧光寿命长（520 micros），为 EDTA-Eu(3+)的时间分辨荧光检测提供了一种新型的解离增强溶液。在第二种方法中，将三元 Eu(3+)配合物，4,4'-双（1'',1'',1'',2'',2'',3'',3''-七氟-4'',6''-己二酮-6''-基）-氯磺基-o-三联苯（BHHCT）-Eu(3+)-DPBT，共价结合到 BSA 上形成水溶性 BSA-BHHCT-Eu(3+)-DPBT 缀合物。这种生物相容性缀合物在水相中具有可见光激发的特征，激发波长范围从紫外光到可见光（最大在 387nm），荧光寿命长（460 micros），量子产率高（27%）。该缀合物成功地用于标记链霉亲和素（SA）和时间分辨荧光成像检测三种环境病原体，即蓝氏贾第鞭毛虫、鼠隐孢子虫和微小隐孢子虫，在水样中的检测。我们的策略为设计用于时间分辨荧光生物分析应用的可见光敏化 Eu(3+)配合物提供了一个通用思路。
