Diffusion free interface

Microseeding attempts to avoid the issue of secondary nucle-ation via fragmentation. In this case, the seed crystal is crushed into many microcrystals on purpose. A set of serial dilutions is conducted until the number of seeds is one or a few within a given aliquot (typically a few microliters). The desirable level of dilution requires some judgement and some screening, but the technique can reproducibly generate a few crystals within a given drop without concern for secondary nucleation. 

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Hansen CL, Skordalakes E, Berger JM, Quake SR (2002) A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion. Proc Natl Acad Sci USA 99 16531-16536 Herzig-Marx R, Queeney KT, Rebecca JJ, Schmidt MA, Jensen KF (2004) Infrared spectroscopy for chemically specific sensing in silicon-based microreactors. Anal Chem 76 6476-6483... 

The liquid-liquid free interface diffusion (FID) method, in which protein and precipitant solutions are carefully superimposed and left to slowly mix diffusively, was least used in the past due to handling difficulties. However, in the last 4 years the free interface technique has experienced a revival for both screening and optimization procedures. The... 

A microfluidic chip has been developed for rapid screening of protein crystallization conditions (Hansen et al., 2002) using the free interface diffusion method. The chip is comprised of a multilayer, silicon elastomer and has 480 valves operated by pressure. The valves are formed at the intersection of two channels separated by a thin membrane. When pressure is applied to the top channel it collapses... 

Conventional free interface diffusion achieves high transient levels of supersaturation, but has a complicated spatial/temporal gradient due to the constant cross-section of the capillary. This gradient couples the kinetics and thermodynamics of traditional free interface diffusion assays in a way that pFID does not. 

Microfluidic Free Interface Diffusion 1 hour Equilibration of Bromophenol Blue... 

Hansen, C. L., et ah, A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion. PNAS 2002, 99, 16531— 16536. 

Salemme, F.R., A free interface diffusion technique for the crystallization of proteins for X-ray crystallography. Arch. Bio-chem. Biophys. 1972, 151. 

FIGURE 2.7 With the free interface diffusion method illustrated schematically here, the protein sample, in buffer, is simply layered, with care, atop the precipitant solution, which may be either salt or polyethylene glycol. Salt ions diffuse rapidly into the protein solution aided by convective transport, and local concentration gradients are created in the region of the interface. With polymeric precipitants, both the polymer and the protein diffuse into one another, but at a greatly reduced rate. 

Through the vapor phase, then, water is removed slowly from the droplet of mother liquor, its pH may be changed, or volatile solvents such as ethanol may be gradually introduced. As with the liquid-liquid dialysis and free-interface diffusion methods, the procedure may be carried out at a number of different temperatures to gain advantage of that parameter as well. 

There are other techniques, however, including microbatch crystallization, where the protein and precipitant are just mixed at the final supersaturation concentration. Free interface diffusion is similar to microbatch but the two components have to diffuse toward each other the concentrations of both protein and precipitant therefore vary with distance from the original interface. In microdialysis, the precipitant solution is allowed to equilibrate with the protein solution through a semipermeable membrane, which permits passage of the precipitant but not the protein (Figure 7). Of these techniques, the first two also lend themselves to automation. 

In addition to the vapor diffusion method described previously, other techniques such as the batch and micro-batch methods, bulk and micro dialysis, free interface diffusion, liquid bridge, and concentration dialysis have also been developed to produce crystals for x-ray diffraction analysis (see McPherson, 1982 and McPherson, 1999). 

Figure 12.20 The free interface diffusion method utilizes a capillary to minimize convective mixing between the protein solution and the second layer of solution containing the crystallization agent (e.g., salt or PEG). The capillary is sealed at both ends with an appropriate adhesive or wax to prevent evaporation of the solution.

Figure 12.21 The ideal trajectory of a free interface diffusion experiment. Each location within the tube undergoes a different trajectory. At one particular location within the capillary, the ideal trajectory will be sampled as depicted above. Note that this ideal trajectory is very similar to Figure 12.19.

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