Bimodal quantitative monitoring for enzymatic activity with simultaneous signal increases in 19F NMR and fluorescence using silica nanoparticle-based molecular probes.
Bimodal quantitative monitoring for enzymatic activity with simultaneous signal increases in 19F NMR and fluorescence using silica nanoparticle-based molecular probes.
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DOI：
10.1021/bc100381x
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发表时间：
2011-07
影响因子：
4.7
通讯作者：
Kazuo Tanaka;Narufumi Kitamura;Y. Chujo
中科院分区：
文献类型：
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作者：
Kazuo Tanaka;Narufumi Kitamura;Y. Chujo
我们描述了利用基于纳米粒子的分子探针在~(19)F核磁共振光谱和荧光光谱中对酶活性进行双峰定量分析。以磷酸盐笼状荧光素为连接物，将全氟树枝状大分子固定在二氧化硅纳米颗粒上。在酶促反应前，全氟化树枝状大分子的分子转动必须受到严格的限制，而且相对于噪声水平，全氟化树状大分子的~(19)F核磁共振信号太宽而无法检测到。荧光素的荧光信号被二磷酸基团的存在所抑制。在碱性磷酸酶作用下，全氟树状大分子和荧光素被释放出来，相应地观察到了全氟树状大分子的核磁共振信号和荧光素的强荧光。从荧光信号和~(19)F-核磁共振信号的增强来检测碱性磷酸酶的酶活性和反应速率。最后，通过对细胞裂解物中酶活性的测定，证明了该探针在仿生条件下存在杂类分子时的可行性。使用(19)F-核磁共振光谱和荧光光谱都可以完成定量分析。
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.