Interfacing Microwells with Nanoliter Compartments: A Sampler Generating High-Resolution Concentration Gradients for Quantitative Biochemical Analyses in Droplets
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- 作者：Fabrice Gielen ; Tomas Buryska ; Liisa Van Vliet ; Maren Butz ; Jiri Damborsky ; Zbynek Prokop ; Florian Hollfelder
- 刊名：Analytical Chemistry
- 出版年：2015
- 出版时间：January 6, 2015
- 年：2015
- 卷：87
- 期：1
- 页码：624-632
- 全文大小：474K
- ISSN：1520-6882
文摘
Analysis of concentration dependencies is key to the quantitative understanding of biological and chemical systems. In experimental tests involving concentration gradients such as inhibitor library screening, the number of data points and the ratio between the stock volume and the volume required in each test determine the quality and efficiency of the information gained. Titerplate assays are currently the most widely used format, even though they require microlitre volumes. Compartmentalization of reactions in pico- to nanoliter water-in-oil droplets in microfluidic devices provides a solution for massive volume reduction. This work addresses the challenge of producing microfluidic-based concentration gradients in a way that every droplet represents one unique reagent combination. We present a simple microcapillary technique able to generate such series of monodisperse water-in-oil droplets (with a frequency of up to 10 Hz) from a sample presented in an open well (e.g., a titerplate). Time-dependent variation of the well content results in microdroplets that represent time capsules of the composition of the source well. By preserving the spatial encoding of the droplets in tubing, each reactor is assigned an accurate concentration value. We used this approach to record kinetic time courses of the haloalkane dehalogenase DbjA and analyzed 150 combinations of enzyme/substrate/inhibitor in less than 5 min, resulting in conclusive Michaelis鈥揗enten and inhibition curves. Avoiding chips and merely requiring two pumps, a magnetic plate with a stirrer, tubing, and a pipet tip, this easy-to-use device rivals the output of much more expensive liquid handling systems using a fraction (鈭?00-fold less) of the reagents consumed in microwell format.