Hirata Junko, de Jong Camiel F, van Dongen Maarten M, Buijs Joost, Ariese Freek, Irth Hubertus, Gooijer Cees
Laser Center, Department of Analytical Chemistry and Applied Spectroscopy, Vrije Universiteit Amsterdam, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Anal Chem. 2004 Aug 1;76(15):4292-8. doi: 10.1021/ac049465o.
A flow injection analysis (FIA) system for biochemical assays using time-resolved fluorescence resonance energy transfer (TR-FRET) in the millisecond time scale was developed. As a model system, we studied a kinase assay, measuring the phosphorylation of poly(GT)-biotin (substrate) by a receptor tyrosine kinase (epidermal growth factor receptor). A streptavidin labeled with XL665 (SA-XL665)-the acceptor-was coupled to the biotin moiety, and an antiphosphotyrosine antibody labeled with europium cryptate (Ab-EuK)-the donor-was coupled to the phosphorylated tyrosine group(s). Long-lived FRET can only occur if the substrate is successfully phosphorylated. For the time-resolved detection of such long-lived luminescence phenomena in a flow system, the repetition rate of the excitation source plays a crucial role. Good results were obtained for a small-sized commercially available quadrupled Nd:YAG laser emitting at 266 nm with a repetition rate of 7.8 kHz and a pulse width of 0.3 ns. The long-lived emissions of the donor at 625 nm and that of the acceptor at 665 nm were monitored simultaneously with two photomultipliers, using a delay time of 50 micros and a gate time of 75 micros to exclude background fluorescence interferences. In the FIA experiments, the Ab-EuK concentration was 6 nM and the substrate concentration and SA-XL665 concentrations were 7 nM. By monitoring the intensity changes at 625 and 665 nm, the inhibition of tyrosine kinase by tyrphostin AG1478 was studied and an IC(50) value of 5.1 +/- 0.4 nM obtained.
开发了一种用于生化分析的流动注射分析（FIA）系统，该系统在毫秒时间尺度上使用时间分辨荧光共振能量转移（TR-FRET）。作为一个模型系统，我们研究了一种激酶分析，即测量受体酪氨酸激酶（表皮生长因子受体）对聚（GT）-生物素（底物）的磷酸化作用。用XL665标记的链霉亲和素（SA-XL665）——受体——与生物素部分偶联，用铕穴状化合物标记的抗磷酸酪氨酸抗体（Ab-EuK）——供体——与磷酸化的酪氨酸基团偶联。只有当底物成功磷酸化时，才会发生长寿命的FRET。对于在流动系统中对这种长寿命发光现象进行时间分辨检测，激发源的重复频率起着至关重要的作用。对于一台小型商用四倍频Nd:YAG激光器，其发射波长为266 nm，重复频率为7.8 kHz，脉冲宽度为0.3 ns，获得了良好的结果。用两个光电倍增管同时监测供体在625 nm处的长寿命发射和受体在665 nm处的长寿命发射，使用50微秒的延迟时间和75微秒的门控时间来排除背景荧光干扰。在FIA实验中，Ab-EuK的浓度为6 nM，底物浓度和SA-XL665的浓度为7 nM。通过监测625和665 nm处的强度变化，研究了酪氨酸激酶抑制剂 tyrphostin AG1478对酪氨酸激酶的抑制作用，并获得了5.1±0.4 nM的IC(50)值。
Zotero 插件