Difference between revisions of "Using microfluidics to decouple nucleation and growth of protein crystals"

Original entry: Dariela Almeda - AP 225, Fall 2009 Article Authors: Jung-uk Shim, Galder Cristobal, Darren R. Link, Todd Thorsen, and Seth Fraden

Keywords

Vapor Diffusion Method, protein nucleation, microfluidics, crystallization.

Summary

This article presented a high throughput, microfluidic device used for protein crystallization. Protein crystallization is required in order discover the 3-D molecular structures of proteins. To crystallize a protein, the sample must be at least 97% pure and must not have crystal defects. Due to the presence of a high energy activation barrier in all protein crystallization processes, a supersaturated protein solution is required to allow formation of crystals. Supersaturating the solution, however, often leads to crystal defects. The current protein crystallization methods including vapor diffusion, interface diffusion, and microbatch have already been implemented in microfluidic devices but the crystallization process still depends on the kinetics of diffusion. Furthermore, microdialysis and crystal seeding are other crystallization methods that allow variation of protein concentration but are not high throughput.

Soft Matter Connection

The microfluidic device, known as the Phase Chip, is made out of polydimethylsiloxane (PDMS) and requires only 1 nL of protein solution. Other methods require 1$\mu$L per experiment. The device consists of wells connected by channels and reservoirs located underneath the wells. A membrane separates the wells from the reservoirs. Protein solution drops are injected into an oil medium in the device's channels. The drops instantly fill in the empty wells and surfactant is added in order to prevent aggregation between drops. When dry air or a salt solution is introduced into the reservoirs, the water in the protein solution permeates through the membrane and into the reservoir. When pure water is introduced into the reservoir, water permeates through the membrane and into the well containing the concentrated protein solution. The diffusion of water is modeled by Fick's First Law:

$J = -D \nabla c = Dc/l$, where J is the flux of water, D is the water diffusion coefficient $(2 x 10^{-9} m^2/s)$, and concentration $c = 30 mol/m^3$.

Protein growth occurs in metastable states that are affected by changes in concentration. By supersaturating the protein solution, the authors are able to create small crystals or "seeds" that nucleate. Once the solution is nucleated, pure water is introduced into the reservoir, which decreases the level of supersaturation in the well and causes the small crystals to dissolve and large crystals to grow. The Phase Chip allows for reversible control of protein concentration as a means to direct nucleation and growth of crystals.

Fig.1 (a) Plan view of the Phase Chip. (b) Vertical section showing the channel, well, and reservoir. (c) Storage of drops in wells guided by surface tension. (d)-(f) Protein crystallization process. Dehydration stage takes about 1 hr with a 1 nL sample. Image of (f) was taken after 5 days of dehydration.