Laterally mobile, functionalized self-assembled monolayers at the fluorous-aqueous interface in a plug-based microfluidic system: characterization and testing with membrane protein crystallization.

This paper describes a method to generate functionalizable, mobile self-assembled monolayers (SAMs) in plug-based microfluidics. Control of interfaces is advancing studies of biological interfaces, heterogeneous reactions, and nanotechnology. SAMs have been useful for such studies, but they are not laterally mobile. Lipid-based methods, though mobile, are not easily amenable to setting up the hundreds of experiments necessary for crystallization screening. Here we demonstrate a method, complementary to current SAM and lipid methods, for rapidly generating mobile, functionalized SAMs. This method relies on plugs, droplets surrounded by a fluorous carrier fluid, to rapidly explore chemical space. Specifically, we implemented his-tag binding chemistry to design a new fluorinated amphiphile, RfNTA, using an improved one-step synthesis of RfOEG under Mitsunobu conditions. RfNTA introduces specific binding of protein at the fluorous-aqueous interface, which concentrates and orients proteins at the interface, even in the presence of other surfactants. We then applied this approach to the crystallization of a his-tagged membrane protein, Reaction Center from Rhodobacter sphaeroides, performed 2400 crystallization trials, and showed that this approach can increase the range of crystal-producing conditions, the success rate at a given condition, the rate of nucleation, and the quality of the crystal formed.

Controlling nonspecific protein adsorption in a plug-based microfluidic system by controlling interfacial chemistry using fluorous-phase surfactants.

Control of surface chemistry and protein adsorption is important for using microfluidic devices for biochemical analysis and high-throughput screening assays. This paper describes the control of protein adsorption at the liquid-liquid interface in a plug-based microfluidic system. The microfluidic system uses multiphase flows of immiscible fluorous and aqueous fluids to form plugs, which are aqueous droplets that are completely surrounded by fluorocarbon oil and do not come into direct contact with the hydrophobic surface of the microchannel. Protein adsorption at the aqueous-fluorous interface was controlled by using surfactants that were soluble in fluorocarbon oil but insoluble in aqueous solutions. Three perfluorinated alkane surfactants capped with different functional groups were used: a carboxylic acid, an alcohol, and a triethylene glycol group that was synthesized from commercially available materials. Using complementary methods of analysis, adsorption was characterized for several proteins (bovine serum albumin (BSA) and fibrinogen), including enzymes (ribonuclease A (RNase A) and alkaline phosphatase). These complementary methods involved characterizing adsorption in microliter-sized droplets by drop tensiometry and in nanoliter plugs by fluorescence microscopy and kinetic measurements of enzyme catalysis. The oligoethylene glycol-capped surfactant prevented protein adsorption in all cases. Adsorption of proteins to the carboxylic acid-capped surfactant in nanoliter plugs could be described by using the Langmuir model and tensiometry results for microliter drops. The microfluidic system was fabricated using rapid prototyping in poly(dimethylsiloxane) (PDMS). Black PDMS microfluidic devices, fabricated by curing a suspension of charcoal in PDMS, were used to measure the changes in fluorescence intensity more sensitively. This system will be useful for microfluidic bioassays, enzymatic kinetics, and protein crystallization, because it does not require surface modification during fabrication to control surface chemistry and protein adsorption.

In situ data collection and structure refinement from microcapillary protein crystallization.

In situ X-ray data collection has the potential to eliminate the challenging task of mounting and cryocooling often fragile protein crystals, reducing a major bottleneck in the structure determination process. An apparatus used to grow protein crystals in capillaries and to compare the background X-ray scattering of the components, including thin-walled glass capillaries against Teflon, and various fluorocarbon oils against each other, is described. Using thaumatin as a test case at 1.8 Å resolution, this study demonstrates that high-resolution electron density maps and refined models can be obtained from in situ diffraction of crystals grown in microcapillaries.

Screening of protein crystallization conditions on a microfluidic chip using nanoliter-size droplets.

Protein crystallization is a major bottleneck in determining tertiary protein structures from genomic sequence data. This paper describes a microfluidic system for screening hundreds of protein crystallization conditions using less than 4 nL of protein solution for each crystallization droplet. The droplets are formed by mixing protein, precipitant, and additive stock solutions in variable ratios in a flow of water-immiscible fluids inside microchannels. Each droplet represents a discrete trial testing different conditions. The system has been validated by crystallization of several water-soluble proteins.