In situ structural analysis with a SAXS laboratory beamline on a microfluidic chip.

Recent advances have been made in coupling microfluidic chips with X-ray equipment, enabling structural analysis of samples directly in microfluidic devices. This important step mainly took place at powerful synchrotron facilities because of the need for a beam reduced in size to fit the microfluidic channel dimensions but still intense. In this work, we discuss how improvements of an X-ray laboratory beamline and an optimal design of a microfluidic device allow reliable structural information to be obtained without the need for a synchrotron. We evaluate the potential of these new developments by probing several well known dispersions. These include dense inorganic gold and silica nanoparticles that scatter photons quite intensely, the bovine serum albumin (BSA) macromolecule, with moderate contrast, to highlight possible applications in biology, and latex nanospheres with only weak contrast with the solvent to show the limits of the setup. We established a proof of concept for a versatile setup that will open the way for more complex lab-on-a-chip devices suitable for in situ and operando structural analysis by small angle X-ray scattering analysis without the necessity for a synchrotron source.

Microfluidics: A Novel Approach for Dehydration Protein Droplets.

The equation of state of colloids plays an important role in the modelling and comprehension of industrial processes, defining the working conditions of processes such as drying, filtration, and mixing. The determination of the equation is based on the solvent equilibration, by dialysis, between the colloidal suspension and a reservoir with a known osmotic pressure. In this paper, we propose a novel microfluidic approach to determine the equation of state of a lysozyme solution. Monodispersed droplets of lysozyme were generated in the bulk of a continuous 1-decanol phase using a flow-focusing microfluidic geometry. In this multiphasic system and in the working operation conditions, the droplets can be considered to act as a permeable membrane system. A water mass transfer flow occurs by molecule continuous diffusion in the surrounding 1-decanol phase until a thermodynamic equilibrium is reached in a few seconds to minutes, in contrast with the standard osmotic pressure measurements. By changing the water saturation of the continuous phase, the equation of state of lysozyme in solution was determined through the relation of the osmotic pressure between protein molecules and the volume fraction of protein inside the droplets. The obtained equation shows good agreement with other standard approaches reported in the literature.

An innovative data processing method for studying nanoparticle formation in droplet microfluidics using X-rays scattering.

X-ray scattering techniques provide a powerful means of characterizing the formation of nanoparticles in solution. Coupling these techniques to segmented-flow microfluidic devices that offer well-defined environments gives access to in situ time-resolved analysis, excellent reproducibility, and eliminates potential radiation damage. However, analysis of the resulting datasets can be extremely time-consuming, where these comprise frames corresponding to the droplets alone, the continuous phase alone, and to both at their interface. We here describe a robust, low-cost, and versatile droplet microfluidics device and use it to study the formation of magnetite nanoparticles with simultaneous synchrotron SAXS and WAXS. Lateral outlet capillaries facilitate the X-ray analysis and reaction times of between a few seconds and minutes can be accommodated. A two-step data processing method is then described that exploits the unique WAXS signatures of the droplets, continuous phase, and interfacial region to identify the frames corresponding to the droplets. These are then sorted, and the background scattering is subtracted using an automated frame-by-frame approach, allowing the signal from the nanoparticles to be isolated from the raw data. Modeling these data gives quantitative information about the evolution of the sizes and structures of the nanoparticles, in agreement with TEM observations. This versatile platform can be readily employed to study a wide range of dynamic processes in heterogeneous systems.

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

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.

Broadcasting photonic lab on a chip concept through a low cost manufacturing approach.

A low cost fabrication process for photonic lab on a chip systems is here proposed. For the implementation of the masters suitable for cast molding fabrication, an inexpensive dry film photoresist, patternable using standard laboratory equipment, is benchmarked against standardized SU-8 masters obtained using UV lithography and systems manufacture in clean room facilities. Results show adequate system fabrication and a comparable performance of the photonic structures for absorbance/extinction measurements.

Coupling High Throughput Microfluidics and Small-Angle X-ray Scattering to Study Protein Crystallization from Solution.

In this work, we propose the combination of small-angle X-ray scattering (SAXS) and high throughput, droplet based microfluidics as a powerful tool to investigate macromolecular interactions, directly related to protein solubility. For this purpose, a robust and low cost microfluidic platform was fabricated for achieving the mixing of proteins, crystallization reagents, and buffer in nanoliter volumes and the subsequent generation of nanodroplets by means of a two phase flow. The protein samples are compartmentalized inside droplets, each one acting as an isolated microreactor. Hence their physicochemical conditions (concentration, pH, etc.) can be finely tuned without cross-contamination, allowing the screening of a huge number of saturation conditions with a small amount of biological material. The droplet flow is synchronized with synchrotron radiation SAXS measurements to probe protein interactions while minimizing radiation damage. To this end, the experimental setup was tested with rasburicase (known to be very sensitive to denaturation), proving the structural stability of the protein in the droplets and the absence of radiation damage. Subsequently weak interaction variations as a function of protein saturation was studied for the model protein lysozime. The second virial coefficients (A2) were determined from the X-ray structure factors extrapolated to the origin. A2 obtained values were found to be in good agreement with data previously reported in literature but using only a few milligrams of protein. The experimental results presented here highlight the interest and convenience of using this methodology as a promising and potential candidate for studying protein interactions for the construction of phase diagrams.