Sébastien Teychené

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.

Structure of the calcium pyrophosphate monohydrate phase (Ca2P2O7·H2O): towards understanding the dehydration process in calcium pyrophosphate hydrates.

Calcium pyrophosphate hydrate (CPP, Ca(2)P(2)O(7) · nH2O) and calcium orthophosphate compounds (including apatite, octacalcium phosphate etc.) are among the most prevalent pathological calcifications in joints. Even though only two dihydrated forms of CPP (CPPD) have been detected in vivo (monoclinic and triclinic CPPD), investigations of other hydrated forms such as tetrahydrated or amorphous CPP are relevant to a further understanding of the physicochemistry of those phases of biological interest. The synthesis of single crystals of calcium pyrophosphate monohydrate (CPPM; Ca(2)P(2)O(7) · H2O) by diffusion in silica gel at ambient temperature and the structural analysis of this phase are reported in this paper. Complementarily, data from synchrotron X-ray diffraction on a CPPM powder sample have been fitted to the crystal parameters. Finally, the relationship between the resolved structure for the CPPM phase and the structure of the tetrahydrated calcium pyrophosphate β phase (CPPT-β) is discussed.

Crystallisation of a highly metastable hydrated calcium pyrophosphate phase

A simple and fast synthesis method was set up to obtain pure hydrated calcium pyrophosphate (CPP) phases of biological interest. This work focused on a specific phase synthesised at 25 °C and pH 4.5 in a stirred tank reactor. Powder X-ray diffraction, FTIR spectroscopy, scanning electron microscopy and thermogravimetric analyses revealed that the phase is unknown but presents similarities with a monoclinic tetrahydrated CPP phase (Ca2P2O7·4H2O, m-CPPT β phase) synthesised under the same conditions of pH and temperature. Characterisation of the unreferenced phase (u-CPP) has been performed, especially to better identify its composition, structure and stability, as well as its possible relation to the m-CPPT β phase or to other hydrated CPP phases.

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.

Influence of pH, Temperature and Impurities on the Solubility of an Active Pharmaceutical Ingredient (API)

Solubility, which defines the liquid /solid equilibrium, is a key parameter to control a crystallization process. This work is focused on the effects of pH, temperature and impurities on the aqueous solubility of an Active Pharmaceutical Ingredient (API).As the API is a weak acid (pKa = 3.7), its solubility increases with the pH. On the basis of the experimental curve of solubility, a model was defined to fit the evolution of the solubility as a function of pH. In the case of this compound, studies revealed a weak influence of the temperature in comparison with the pH. So, the solubility of the compound is slightly impacted by the temperature.Some experiments were carried out in order to compare the solubility of the API, at the same pH and temperature, for different concentrations of impurities found in the process. The results revealed a solubility increase in presence of acetic acid and a high solubility decrease in presence of sodium chloride. By carrying out experiments on common ions salts, the anion chloride Cl- has been identified as the cause of the solubility decrease.

Ultra-fast precipitation of transient amorphous cerium oxalate in concentrated nitric acid media

Amorphous cerium oxalate is characterized and reported here for the first time as a primary nucleating transient precursor for a more stable crystalline hydrated phase, at high supersaturation and in strong acid solutions. Preliminary results point out an initial binodal phase separation leading to precipitation.

Coupling microfluidics and SAXS to study the whole crystallization process

Sébastien Teychené1, Dimitri Radajewski1, Nhat Van Pham1, Isaac Rodriguez-Ruiz2, Petra Pernot3, Martha Brennich3, Thomas Bizien4, Béatrice Biscans1, Francoise Bonneté5 1Laboratoire De Génie Chimique University Of Toulouse, Toulouse, France, 2CEA,DEN,DMRC,SA2i, Bagnol-sur-Cèse, France, 3European Synchrotron Radiation Facility, Grenoble, France, 4SWING beamline, Synchrotron Soleil, Gif sur Yvette, France, 5Institut des Biomolécules MaxMousseron, UMR 5247, Université d’Avignon, Avignon, France E-mail: sebastien.teychene@ensiacet.fr

Unstable amorphous cerium oxalate precipitation in concentrated HNO3 media

Rare earths precipitation using oxalic acid is broadly used in the industry of nuclear wastes reprocessing. The selective recovery of actinides from highly concentrated solutions is performed by oxalate salts precipitation in nitric acid media, in the range of 1-7 mol L-1[1]. As lanthanides and actinides in solution display close thermodynamic properties, the first ones are widely used as models for the understanding and development of new processes involving actinides of interest thus avoiding tedious manipulation of radioactive species. Hence, in the abovementioned framework, Ce (III) cations are frequently used in order to minimize the amount of radioactive materials involved in R&D studies. It is known that the precipitation reaction of Ce in HNO3 media leads to the formation of Ce2(C2O4)3·10H2O at equilibrium. Nevertheless, very little has been investigated regarding the system out-of-equilibrium evolution during the initial stages of reaction, and the understanding of the mechanisms involved in cerium oxalate (CeOX) precipitation/nucleation are still poorly understood. With a view to elucidate these mechanisms, CeOX precipitation experiments have been performed at room temperature in small volumes using two different experimental setups, both allowing in-situ monitoring of precipitation mechanisms: a Hele-Shaw cell under optical microscopy for micro-droplet batch precipitation, and an hybrid kapton-OSTEMER microfluidic platform, coupled to small angle X-ray scattering (SAXS), for continuous reaction, using a layer of water as a buffer between reagents to achieve diffusion-driven precipitation. Due to the stochastic nature of nucleation, the use of small volumes allowed us to locate the nucleation events in space, facilitating their observation [2]. The new experimental results presented here are questioning regarding CeOX precipitation mechanisms: SAXS experiments complemented by Monte Carlo simulations [3] highlighted the appearance of disperse spherical particles, of initial size close to 5 nm, which rapidly grow up to larger objects, observable by optical microscopy during the first second of reaction. Complementary characterization using HRSEM and TEM revealed the instantaneous primary precipitation of an initially liquid-like amorphous precipitate, which subsequently undergoes a solventmediated phase transition to a more stable crystalline phase.