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

Abstract

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 Ϸ140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in Ϸ10-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 l of protein solution, Ϸ1,300 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.droplets ͉ plugs ͉ protein structure ͉ high-throughput ͉ miniaturization T his work reports a ''hybrid'' microfluidic approach that uses nanoliter plugs to perform screening and optimization simultaneously in the same experiment. To validate this method using a challenging problem, we demonstrate its compatibility with crystallization of membrane proteins. Small-scale screening and optimization experiments are important for biological assays, chemical screening, and protein crystallization (1-3). Screening and optimization are usually carried out sequentially. In the case of protein crystallization, random sparse matrix screening initially identifies the precipitants that may lead to crystallization. Subsequent gradient optimization establishes concentrations of these precipitants that lead to diffractionquality crystals (4). Combining screening and optimization steps into a single hybrid experiment would eliminate the need to wait for the outcome of the initial screen before carrying out subsequent optimizations. Furthermore, a hybrid experiment would reduce the false negatives (5) associated with screens performed at a single concentration. The hybrid experiment could also be more conclusive, because a single batch of the s...

Using Three-Phase Flow of Immiscible Liquids To Prevent Coalescence of Droplets in Microfluidic Channels: Criteria To Identify the Third Liquid and Validation with Protein Crystallization

“…Membrane proteins are solubilized using detergents, and their crystallization in plugs requires special handling. 47 We did not demonstrate the use of third liquids for crystallization of membrane proteins, because the necessary detailed characterization of potential problems such as detergents being extracted by the third liquid is beyond the scope of this paper.…”

Reaching One Single and Stable Critical Cluster through Finite-Sized Systems

“…But as we have said, volume will be directly related to solubility of the material, so that the range of volumes where confinement occurs for materials other than the lysozyme presented here cannot be predicted. However, technologies such as microfluidics, [24][25][26][27] microfabricated arrays, 28 emulsions, 29,30 and nanoscale "dispensers" 31,32 allow a great range of volumes to be generated. These technologies could offer ways to take advantage of here presented results.…”

Three-Dimensional Nanocrystal Superlattices Grown in Nanoliter Microfluidic Plugs

“…The microfluidic PDMS-devices ( Figure S1) with four or five inputs were fabricated as described in Ref. [6][7][8] The microchannel surface of PDMS-device was functionalized first with (tridecafluoro-1,1,2,2-tetrahydrooctyl)-trichlorosilane and then coated with with amorphous fluoropolymer solution (Poly(4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-cotetrafluoroethylene)), 9 to make PDMS-surface hydrophobic and compatible with organic solvents.…”

“…8 were floated out onto silicon substrate and studied with scanning electron microscopy (SEM). For kinetic studies, to overcome the problem of plugs moving and merging we prepared the plugs with elongated shape.…”