Using nanoliter plugs in microfluidics to facilitate and understand protein crystallization

Abstract

Protein crystallization is important for determining protein structures by X-ray diffraction. Nanoliter-sized plugs--aqueous droplets surrounded by a fluorinated carrier fluid--have been applied to the screening of protein crystallization conditions. Preformed arrays of plugs in capillary cartridges enable sparse matrix screening. Crystals grown in plugs inside a microcapillary may be analyzed by in situ X-ray diffraction. Screening using plugs, which are easily formed in PDMS microfluidic channels, is simple and economical, and minimizes consumption of the protein. This approach also has the potential to improve our understanding of the fundamentals of protein crystallization, such as the effect of mixing on the nucleation of crystals.

Figure 1

Schematic illustration of protein crystals in aqueous plugs surrounded by fluorinated carrier fluid inside a glass capillary.

Figure 2

Sparse matrix screening is performed using preformed cartridges and a microfluidic T-junction. (a) A preformed cartridge of plugs containing different precipitants merges with a stream of protein solution at a T-junction (top). The resulting array of plugs is transferred into a receiving capillary, the flow is stopped, the array is incubated and the optimal precipitant yields a protein crystal (bottom). (b) Photograph of a T-junction microfluidic device. Two funnel-shaped glass capillaries are used to couple the cartridge to the inlet and the receiving capillary to the outlet of the microfluidic channel.

Figure 3

Optimization screens are performed by varying the flow rates of aqueous streams of crystallization reagents. (a) Microphotograph showing the microfluidic device for generating aqueous plugs. There are three inlets for aqueous streams and one inlet for carrier fluid. The receiving capillary is inserted into the outlet channel. The entire chip is approximately 1 × 1 inch. The background of the image was brightened in Photoshop for clarity. (b) When the flow rate of the precipitant (blue) is high, the concentration of the precipitant in the plug is also high. (c) As the flow rate of the precipitant is decreased and the flow rate of the buffer is increased, the concentration of the precipitant decreases accordingly.

Figure 4

Schematic drawing showing the process of water transfer from a plug containing a protein and a precipitant to a plug containing a desiccant solution of higher osmotic pressure. The carrier fluid is water permeable. After incubation, the plug containing protein and precipitant will be concentrated, and could yield protein crystals.

Figure 5

In situ diffraction of a thaumatin crystal at 1.8 Å resolution. A crystal was grown inside a plug in a capillary. The capillary was mounted on a goniometer and the crystal, while still in the plug, was subjected to the X-ray beam to obtain the diffraction pattern. The diffraction pattern is comparable to those obtained from crystals grown using conventional methods. The thaumatin crystal was ~100 × 100 μm and data were collected at Argonne National Laboratory’s BioCARS beamline sector 14.

Figure 6

Control of mixing can lead to control of nucleation in protein crystallization. (a) Schematic illustration of the plug-based microfluidic setup for studying the effect of mixing on the nucleation of protein crystals. Mixing by chaotic advection is illustrated. Microphotographs showing an example of the effect of (b) slow chaotic mixing and (c) fast chaotic mixing on protein crystallization under otherwise identical conditions. The diameter of the receiving capillary is 200 μm.