Studying reaction kinetics by simultaneous FRET and cross-correlation analysis in a miniaturized continuous flow reactor
Studying reaction kinetics by simultaneous FRET and cross-correlation analysis in a miniaturized continuous flow reactor
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DOI：
10.1039/b406899h
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发表时间：
2004-01-01
影响因子：
3.3
通讯作者：
Schwille, P
中科院分区：
文献类型：
--
作者：
Dittrich, PS;Müller, B;Schwille, P
在这项研究中，我们提出了一种改进的光学检测技术的基础上共聚焦荧光光谱在小型化连续流（muCF）反应器的快速反应的调查。特殊的设置允许同时观察反应的基础上的荧光共振能量转移（FRET）作为反应进程的指标。通过空间荧光互相关确定流速使得能够将空间信息（即检测点的位置）转换成相应的时间信息。克服了传统连续式的缺点。在高样品消耗的低反应器中，我们使用微流控芯片，其允许低浓度样品溶液的经济消耗，而硅/玻璃芯片完全适合于共聚焦设置。在这些通道内，通过将溶液挤压成薄层，在三个通道的交叉处进行层流条件下的快速、基于扩散的混合。使用该mCF装置，我们研究了由酶核酸外切酶III引起的双链DNA寡聚体的不可逆切割反应。互补DNA链分别用塔姆拉和Cy 5染料分子染色，如果两条链退火，则进行能量转移。Cy 5荧光的减少直接对应于裂解反应的进展。
In this study we present a refined optical detection technique for investigation of fast reactions based on confocal fluorescence spectroscopy in a miniaturized continuous flow (muCF) reactor. The special setup allows for simultaneous observation of the reaction on the basis of fluorescent resonance energy transfer (FRET) as an indicator of the reaction progress. Determination of the flow velocity via spatial fluorescence cross-correlation enables the conversion of spatial information, i.e. the position of the detection point, into the respective temporal information. To overcome the disadvantage of conventional continuous. ow reactors of high sample consumption, we used a microfluidic chip allowing an economical expenditure of sample solution at low concentration, while the silicon/glass chip is perfectly adaptable to the confocal setup. Inside these channels, rapid, diffusion-based mixing under laminar flow conditions is performed at the crossing of three channels by squeezing the solutions into thin layers. Using this mCF device, we investigated the irreversible cleavage reaction of a double stranded DNA oligomer by the enzyme exonuclease III. The complementary DNA strands are stained with TAMRA and Cy 5 dye molecules, respectively, undergoing an energy transfer if both strands are annealed. The reduction of the Cy 5 fluorescence directly corresponds to the progress of the cleavage reaction.