Using Microfluidics to Decouple Nucleation and Growth of Protein Crystals

Abstract

A high throughput, low volume microfluidic device has been designed to decouple the physical processes of protein crystal nucleation and growth. This device, called the Phase Chip, is constructed out of poly(dimethylsiloxane) (PDMS) elastomer. One of the Phase Chip's innovations is to exploit surface tension forces to guide each drop to a storage chamber. We demonstrate that nanoliter water-in-oil drops of protein solutions can be rapidly stored in individual wells thereby allowing the screening of 1000 conditions while consuming a total of only 10 mug protein on a 20 cm(2) chip. Another significant advance over current microfluidic devices is that each well is in contact with a reservoir via a dialysis membrane through which only water and other low molecular weight organic solvents can pass, but not salt, polymer, or protein. This enables the concentration of all solutes in a solution to be reversibly, rapidly, and precisely varied in contrast to current methods, such as the free interface diffusion or sitting drop methods, which are irreversible. The Phase Chip operates by first optimizing conditions for nucleation by using dialysis to supersaturate the protein solution, which leads to nucleation of many small crystals. Next, conditions are optimized for crystal growth by using dialysis to reduce the protein and precipitant concentrations, which leads small crystals to dissolve while simultaneously causing only the largest ones to grow, ultimately resulting in the transformation of many small, unusable crystals into a few large ones.

Figure 1

(a) Plan view of the Phase Chip. One reservoir (red) located underneath 100 wells (green circles) is shown here. There are five such sections on the chip. (b) Vertical section of a storage well, channel, reservoir and dialysis membrane. The device is constructed from two PDMS layers and subsequently sealed together (Squires and Quake 2005). In the upper, thick (5 mm) layer, there are flow channels and storage wells. In the lower, thin (40 μm) layer, there is a reservoir, sealed by a 15 μm thick PDMS membrane, The reservoir is formed by spin coating a 40 μm thick layer PDMS over a 25 μm high photoresist mold (Squires and Quake 2005). (c) Photographs of surface tension guided storage of aqueous drops into rectangular wells, without a reservoir. (d-f) Protein crystallization with reversible dialysis. The photographs are of a single 300 μm diameter circular well that contains protein solution. The channel is on the right side of the well. The square posts are 30 μm wide and support a 15 μm thick PDMS membrane, which forms the bottom of the well. (d) A stable protein solution of xylanase slightly overfills the well. (e) Protein gelation occurred after the reservoir was filled with 5M NaCl. (f) The reservoir was filled with pure water, which rehydrated the precipitate, transforming them into crystals.