Using microfluidics to observe the effect of mixing on nucleation of protein crystals

This paper analyzes the effect of mixing on nucleation of protein crystals. The mixing of protein and precipitant was controlled by changing the flow rate in a plug-based microfluidic system. The nucleation rate inversely depended on the flow rate, and flow rate could be used to control nucleation. For example, at higher supersaturations, precipitation happened at low flow rates while large crystals grew at high flow rates. Mixing at low flow velocities in a winding channel induces nucleation more effectively than mixing in straight channels. A qualitative scaling argument that relies on a number of assumptions is presented to understand the experimental results. In addition to helping fundamental understanding, this result may be used to control nucleation, using rapid chaotic mixing to eliminate formation of precipitates at high supersaturation and using slow chaotic mixing to induce nucleation at lower supersaturation

Salvage and storage of infectious disease protein targets in the SSGCID high-throughput crystallization pathway using microfluidics

SSGCID protein crystals were salvaged and stored using the MPCS Plug Maker and CrystalCards when high-throughput traditional sitting-drop vapor diffusion initially failed

Microfluidic Systems for Chemical Kinetics that Rely on Chaotic Mixing in Droplets

This paper reviews work on a microfluidic system that relies on chaotic advection to rapidly mix multiple reagents isolated in droplets (plugs). Using a combination of turns and straight sections, winding microfluidic channels create unsteady fluid flows that rapidly mix the multiple reagents contained within plugs. The scaling of mixing for a range of channel widths, flow velocities and diffusion coefficients has been investigated. Due to rapid mixing, low sample consumption and transport of reagents with no dispersion, the system is particularly appropriate for chemical kinetics and biochemical assays. The mixing occurs by chaotic advection and is rapid (sub-millisecond), allowing for an accurate description of fast reaction kinetics. In addition, mixing has been characterized and explicitly incorporated into the kinetic model

Experimental test of scaling of mixing by chaotic advection in droplets moving through microfluidic channels

This letter describes an experimental test of a simple argument that predicts the scaling of chaotic mixing in a droplet moving through a winding microfluidic channel. Previously, scaling arguments for chaotic mixing have been described for a flow that reduces striation length by stretching, folding, and reorienting the fluid in a manner similar to that of the baker’s transformation. The experimentally observed flow patterns within droplets (or plugs) resembled the baker’s transformation. Therefore, the ideas described in the literature could be applied to mixing in droplets to obtain the scaling argument for the dependence of the mixing time, t ∼ (aw/U)log(Pe), where w [m] is the cross-sectional dimension of the microchannel, a is the dimensionless length of the plug measured relative to w, U [m s^−1] is the flow velocity, Pe is the Péclet number (Pe = wU/D), and D [m^2 s^−1] is the diffusion coefficient of the reagent being mixed. Experiments were performed to confirm the scaling argument by varying the parameters w, U, and D. Under favorable conditions, submillisecond mixing has been demonstrated in this system

The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS)

The Microcapillary Protein Crystallization System (MPCS) is a new protein-crystallization technology used to generate nanolitre-sized crystallization experiments for crystal screening and optimization. Using the MPCS, diffraction-ready crystals were grown in the plastic MPCS CrystalCard and were used to solve the structure of methionine-R-sulfoxide reductase

The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS)

This is the published version. Copyright International Union of CrystallographyThe Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, ~10-20 nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one com­prehensive `hybrid' crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants

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 angstrom 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

Time-Controlled Microfluidic Seeding in nL-Volume Droplets To Separate Nucleation and Growth Stages of Protein Crystallization

This paper describes a method of time-controlled seeding to separate the stages of nucleation and growth in protein crystallization using a microfluidic device

Nanovolume optimization of protein crystal growth using the microcapillary protein crystallization system

The Microcapillary Protein Crystallization System (MPCS) is used to successfully optimize protein crystals from 28 out of 29 tested proteins. Six protein structures have been determined from diffraction-ready crystals grown inside and harvested directly from the MPCS CrystalCards, which are compatible with the recently commercialized and automated MPCS Plug Maker instrument