Millisecond kinetics on a microfluidic chip using nanoliters of reagents
Millisecond kinetics on a microfluidic chip using nanoliters of reagents
复制标题
DOI：
10.1021/ja0354566
复制
发表时间：
2003-11-26
影响因子：
15
通讯作者：
Ismagilov, RF
中科院分区：
文献类型：
--
作者：
Song, H;Ismagilov, RF
本文介绍了一种微流控芯片进行动力学测量优于毫秒级的分辨率。在微流控系统中的快速动力学测量由于两个问题而变得复杂：混合慢和分散大。这些问题也使在微流控芯片中进行的生物化学测定复杂化。我们最近显示（宋，H.; Tice，J.D.;伊斯马吉洛夫河F. Angew.化学成分：Int.Ed.2003，42，768-772）如何可以使用微通道中的多相流体流来通过在由不混溶流体包围的水性液滴（塞）内输送试剂来解决这两个问题。在这里，这种基于液滴的微流控系统用于提取酶促反应的动力学参数。核糖核酸酶A（RNase A）的快速单周转动力学测量优于毫秒分辨率使用亚微升体积的溶液。为了获得单次转换速率常数（k = 1100 +/- 250 s（-1）），证明了该微流体平台的四个新特征：（i）快速芯片上稀释，（ii）多时间范围访问，（iii）与RNase A的生物相容性，以及（iv）明确处理混合以提高系统的时间分辨率。这些特点进行了讨论使用RNase A的动力学。从积分2-4秒的荧光图像，可以使用小于150 nL的试剂溶液获得每个动力学曲线，因为该系统依赖于移动液滴内部的混沌平流而不是湍流来实现快速混合。在PDMS中制造这些器件是简单的，除了具有CCD相机的标准显微镜之外，不需要专门的设备来运行实验。这种微流控平台可以作为停流方法的廉价和经济的补充，用于化学和生物化学中广泛的时间分辨实验和测定。
This paper describes a microfluidic chip for performing kinetic measurements with better than millisecond resolution. Rapid kinetic measurements in microfluidic systems are complicated by two problems: mixing is slow and dispersion is large. These problems also complicate biochemical assays performed in microfluidic chips. We have recently shown (Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2003, 42, 768-772) how multiphase fluid flow in microchannels can be used to address both problems by transporting the reagents inside aqueous droplets (plugs) surrounded by an immiscible fluid. Here, this droplet-based microfluidic system was used to extract kinetic parameters of an enzymatic reaction. Rapid single-turnover kinetics of ribonuclease A (RNase A) was measured with better than millisecond resolution using sub-microliter volumes of solutions. To obtain the single-turnover rate constant (k = 1100 +/- 250 s(-1)), four new features for this microfluidics platform were demonstrated: (i) rapid on-chip dilution, (ii) multiple time range access, (iii) biocompatibility with RNase A, and (iv) explicit treatment of mixing for improving time resolution of the system. These features are discussed using kinetics of RNase A. From fluorescent images integrated for 2-4 s, each kinetic profile can be obtained using less than 150 nL of solutions of reagents because this system relies on chaotic advection inside moving droplets rather than on turbulence to achieve rapid mixing. Fabrication of these devices in PDMS is straightforward and no specialized equipment, except for a standard microscope with a CCD camera, is needed to run the experiments. This microfluidic platform could serve as an inexpensive and economical complement to stopped-flow methods for a broad range of time-resolved experiments and assays in chemistry and biochemistry.