In situ microfluidic dialysis for biological small-angle X-ray scattering

Abstract

Owing to the demand for low sample consumption and automated sample changing capabilities at synchrotron small-angle X-ray (solution) scattering (SAXS) beamlines, X-ray microfluidics is receiving continuously increasing attention. Here, a remote-controlled microfluidic device is presented for simultaneous SAXS and ultraviolet absorption measurements during protein dialysis, integrated directly on a SAXS beamline. Microfluidic dialysis can be used for monitoring structural changes in response to buffer exchange or, as demonstrated, protein concentration. By collecting X-ray data during the concentration procedure, the risk of inducing protein aggregation due to excessive concentration and storage is eliminated, resulting in reduced sample consumption and improved data quality. The proof of concept demonstrates the effect of halted or continuous flow in the microfluidic device. No sample aggregation was induced by the concentration process at the levels achieved in these experiments. Simulations of fluid dynamics and transport properties within the device strongly suggest that aggregates, and possibly even higher-order oligomers, are preferentially retained by the device, resulting in incidental sample purification. Hence, this versatile microfluidic device enables investigation of experimentally induced structural changes under dynamically controllable sample conditions.

Preparing monodisperse macromolecular samples for successful biological small-angle X-ray and neutron-scattering experiments

“…A single SAXS measurement typically requires 5–30 μl of sample (50–100 μl for SEC-SAXS, Box 1), with these volumes likely to decrease as microfluidic technologies are introduced59,60. However, what volume can be measured and what volume is required for reproducible experiments may be quite different.…”

A microfluidic device for both on-chip dialysis protein crystallization and in situ X-ray diffraction

“…In our device, crystallization of the proteins occurs under static conditions regarding the protein solution and not under flowing conditions. 40 This is a major asset as crystallizing the protein solutions under flowing conditions can be challenging and can affect the quality of the growing crystals. 42,43 However, the crystallization solution can flow continuously within the microfluidic channel of our device, offering the possibility to exchange dynamically crystallization conditions while using the same protein sample, thus reducing also the consumption of the valuable protein sample.…”

“…30 Commercially available membranes can be employed like regenerated cellulose (RC) dialysis membranes which are widely used in the crystallization of biological macromolecules. A microfluidic setup including two separate modules, a "dialysis chip" and an "exposure chip" for in situ SAXS data collection, was reported by Skou et al 40 In their dialysis chip, an RC membrane is sandwiched in-between two PDMS sheets, clamped together using two pieces of 5 mmthick PMMA plates. Such a dialysis chip was used to concentrate a flowing protein solution using an imposed osmotic pressure difference that drives a solvent flow through the semi-permeable membrane.…”

Microfluidic dialysis using photo-patterned hydrogel membranes in PDMS chips

“…But it is in the field of analytical biochemistry that this type of technique is of particular interest with numerous applications such as on-chip microdialysis sample cleanup, fractionation, concentration, separation, etc, see e.g. [12][13][14][15][16][17] and the review [18].…”