A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale
A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale
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DOI：
10.1016/s0006-3495(98)77977-9
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发表时间：
1998-05-01
影响因子：
3.4
通讯作者：
Roder, H
中科院分区：
文献类型：
--
作者：
Shastry, MCR;Luck, SD;Roder, H
基于Regenfuss等人的原始设计的连续流动毛细混合装置。(Regenfuss，P.，R.M.克莱格，M.J.Fulwyler，F.J.Barrantes，T.M.Jovin。1985年。科西牧师。Instrum。56：283-290)，在混合器设计、检测方法和数据分析方面取得了重大进展。为了克服原始设计中用于观察的自由流动射流的相关问题(不稳定、散射造成的光学伪影、几何清晰度不佳)，从毛细管中出来的溶液被直接注入通过磨玻璃接头连接到外部毛细管尖端的流动池中。采用汞(Xe)弧灯作为激发光源，用带有紫外光敏化的CCD探测器的数码相机进行检测，测量了混合器下游的荧光随距离变化的反应动力学。涉及荧光染料的测试反应表明，混合在开始的15亩S内完成，测量的死时间为45+/-5亩S，这意味着时间分辨率比传统的停流仪提高了30倍。Ccd相机的高灵敏度和线性度有助于在从45亩S到几毫秒的时间窗口内获得无伪影的运动数据，其信噪比水平与传统方法相当。讨论了该方法的适用范围，并以蛋白质折叠反应为例进行了说明。
A continuous-flow capillary mixing apparatus, based on the original design of Regenfuss et al. (Regenfuss, P., R. M. Clegg, M. J. Fulwyler, F. J. Barrantes, and T. M. Jovin. 1985. Rev. Sci. Instrum. 56:283-290), has been developed with significant advances in mixer design, detection method and data analysis. To overcome the problems associated with the free-flowing jet used for observation in the original design (instability, optical artifacts due to scattering, poor definition of the geometry), the solution emerging from the capillary is injected directly into a flow-cell joined to the tip of the outer capillary via a ground-glass joint. The reaction kinetics are followed by measuring fluorescence versus distance downstream from the mixer, using an Hg(Xe) are lamp for excitation and a digital camera with a UV-sensitized CCD detector for detection. Test reactions involving fluorescent dyes indicate that mixing is completed within 15 mu s of its initiation and that the dead time of the measurement is 45 +/- 5 mu s, which represents a >30-fold improvement in time resolution over conventional stopped-flow instruments. The high sensitivity and linearity of the CCD camera have been instrumental in obtaining artifact-free kinetic data over the time window from similar to 45 mu s to a few milliseconds with signal-to-noise levels comparable to those of conventional methods. The scope of the method is discussed and illustrated with an example of a protein folding reaction.