Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Bioconjug Chem. 2011 Aug 17;22(8):1484-90. doi: 10.1021/bc100381x. Epub 2011 Jul 11.
We describe the bimodal quantitative assay for enzymatic activity in (19)F NMR spectroscopy and fluorescence spectroscopy using a nanoparticle-based molecular probe. Perfluorinated dendrimers were tethered on silica nanoparticles with a phosphate-caged fluorescein as a linker. Before enzymatic reaction, the molecular rotation of the perfluorinated dendrimers should be highly restricted, and the (19)F NMR signals from the perfluorinated dendrimers were too broad to be detected relative to the noise level. Fluorescence signals of fluorescein were suppressed by the presence of the diphosphate groups. Following the enzymatic reaction with an alkaline phosphatase, perfluorinated dendrimers and fluorescein were released, and the NMR signals of perfluorinated dendrimers and strong fluorescence from fluorescein were correspondingly observed. The enzymatic activity and reaction rates of the hydrolysis of alkaline phosphatase were detected from the increases of fluorescence and (19)F NMR signals. Finally, the feasibility of the probe in the presence of miscellaneous molecules under biomimetic conditions was demonstrated by determining of the enzymatic activity in cell lysate. Quantitative analysis using both (19)F NMR spectroscopy and fluorescence spectroscopy can be accomplished.
我们描述了一种基于纳米粒子的分子探针，通过（19）F NMR 光谱和荧光光谱进行酶活性的双模态定量分析。全氟代树状大分子通过磷酸酯封端的荧光素作为连接物键合到硅纳米粒子上。在酶反应之前，全氟代树状大分子的分子旋转应该受到高度限制，并且（19）F NMR 信号来自全氟代树状大分子太宽，无法与噪声水平相比检测到。荧光素的荧光信号被二磷酸基团的存在所抑制。在用碱性磷酸酶进行酶反应后，释放出全氟代树状大分子和荧光素，相应地观察到全氟代树状大分子的 NMR 信号和荧光素的强荧光。通过荧光和（19）F NMR 信号的增加来检测碱性磷酸酶水解的酶活性和反应速率。最后，通过在仿生条件下确定细胞裂解物中的酶活性，证明了探针在存在多种分子时的可行性。可以完成使用（19）F NMR 光谱和荧光光谱的定量分析。
