Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 103; no. 51; pp. 19243 - 19248
• Main Authors: Li, Liang, Mustafi, Debarshi, Fu, Qiang, Tereshko, Valentina, Chen, Delai L, Tice, Joshua D, Ismagilov, Rustem F
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 19.12.2006; National Acad Sciences
• Subjects: Biochemistry; Crystallization; Crystallization - methods; Crystals; Detergents; Evaporation; Flow velocity; Membrane proteins; Membrane Proteins - chemistry; Membranes; Microfluidic Analytical Techniques - instrumentation; Microfluidic Analytical Techniques - methods; Models, Molecular; Molecular biology; Physical Sciences; Porins; Proteins; Reagents; Viscosity; X-Ray Diffraction
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0607502103

High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a "hybrid" droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as [almost equal to]140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in [almost equal to]10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 μl of protein solution, [almost equal to]1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.