An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments
An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments
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DOI：
10.1529/biophysj.107.117689
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发表时间：
2008-03-01
影响因子：
3.4
通讯作者：
Puglisi, Joseph D.
中科院分区：
文献类型：
--
作者：
Aitken, Colin Echeverria;Marshall, R. Andrew;Puglisi, Joseph D.
单分子荧光技术在复杂生物体系中的应用对单荧光团的性能提出了更高的要求。为了提高单分子实验中染料的稳定性，我们提出了一种酶促氧清除系统。我们将先前描述的原儿茶酸/原儿茶酸-3，4-双加氧酶系统与目前使用的葡萄糖氧化酶/过氧化氢酶系统进行了比较。在标准条件下，原儿茶酸/原儿茶酸-3，4-双加氧酶体系的溶解氧浓度较低。此外，我们观察到单个Cy3、Cy5和Alexa488荧光团的初始寿命增加。我们进一步测试了化学添加剂在该体系中的作用。我们发现，生物还原剂增加了Cy5的眨眼事件的频率和持续时间，这种影响随着还原潜力的增加而增加。我们观察到Cy3和Alexa488在抗氧化剂抗坏血酸和没食子酸正丙酯存在下的稳定性增加。这种新型的O2-清除体系在单分子荧光实验中具有广阔的应用前景。
The application of single-molecule fluorescence techniques to complex biological systems places demands on the performance of single fluorophores. We present an enzymatic oxygen scavenging system for improved dye stability in single-molecule experiments. We compared the previously described protocatechuic acid/protocatechuate-3,4-dioxygenase system to the currently employed glucose oxidase/catalase system. Under standardized conditions, we observed lower dissolved oxygen concentrations with the protocatechuic acid/protocatechuate-3,4-dioxygenase system. Furthermore, we observed increased initial lifetimes of single Cy3, Cy5, and Alexa488 fluorophores. We further tested the effects of chemical additives in this system. We found that biological reducing agents increase both the frequency and duration of blinking events of Cy5, an effect that scales with reducing potential. We observed increased stability of Cy3 and Alexa488 in the presence of the antioxidants ascorbic acid and n-propyl gallate. This new O-2-scavenging system should have wide application for single-molecule fluorescence experiments.