Innovative High-Throughput SAXS Methodologies Based on Photonic Lab-on-a-Chip Sensors: Application to Macromolecular Studies

Abstract

The relevance of coupling droplet-based Photonic Lab-on-a-Chip (PhLoC) platforms and Small-Angle X-Ray Scattering (SAXS) technique is here highlighted for the performance of high throughput investigations, related to the study of protein macromolecular interactions. With this configuration, minute amounts of sample are required to obtain reliable statistical data. The PhLoC platforms presented in this work are designed to allow and control an effective mixing of precise amounts of proteins, crystallization reagents and buffer in nanoliter volumes, and the subsequent generation of nanodroplets by means of a two-phase flow. Spectrophotometric sensing permits a fine control on droplet generation frequency and stability as well as on concentration conditions, and finally the droplet flow is synchronized to perform synchrotron radiation SAXS measurements in individual droplets (each one acting as an isolated microreactor) to probe protein interactions. With this configuration, droplet physic-chemical conditions can be reproducibly and finely tuned, and monitored without cross-contamination, allowing for the screening of a substantial number of saturation conditions with a small amount of biological material. The setup was tested and validated using lysozyme as a model of study. By means of SAXS experiments, the proteins gyration radius and structure envelope were calculated as a function of protein concentration. The obtained values were found to be in good agreement with previously reported data, but with a dramatic reduction of sample volume requirements compared to studies reported in the literature.

OSTE+ for in situ SAXS analysis with droplet microfluidic devices

“…To overcome this limitation, Pham et al synchronized the acquisition to the passing droplets to study the impact of NaCl concentration on lysozyme in solution. [10,11] The microfluidic droplet generation device was made from thiol-ene (TE) polymer (Norland Optical Adhesive, NOA 81) coupled to a quartz capillary. The same year, Saldanha et al studied the transformation of rod-shaped vimentin into filaments upon salt addition, using a PDMS microfluidic device connected to a quartz capillary.…”

SAS-cam: a program for automatic processing and analysis of small-angle scattering data

“…The recent progress in synchrotron facilities and SAXS instruments has made SAXS data collection highly automated with high-throughput operations (Hura et al, 2009;Blanchet & Svergun, 2013;Liu et al, 2018). This trend is reflected in the fast-growing application of the SAXS method in characterizing the structures of homogeneous biological macromolecules in solution (Rodríguez-Ruiz et al, 2017) and heterogenous molecular systems (Kim et al, 2020).…”

Microfluidic devices for small-angle neutron scattering

“…While a number of microfabrication techniques for microfluidic SAXS (small-angle X-ray scattering) (Greaves & Manz, 2005;Ghazal et al, 2016) have been developed (Shastry et al, 1998;Stehle et al, 2013;Merlin et al, 2011;Møller et al, 2013;Skou et al, 2014;With et al, 2014;Beuvier et al, 2015;Rodríguez-Ruiz et al, 2017), these are not directly transposable to neutrons. Neutrons interact with the atomic nucleus itself, which varies significantly between elements and isotopes.…”