Crystal Nucleation On a Chip – CNOC

* Droplet-based microfluidics:. A dispersed phase is created by mixing several miscible compounds which is then periodically separated by a continuous phase, generating monodisperse droplets. In such devices, droplets are suspended in an external carrier phase so they behave as microreactors. The immiscibility of the two phases prevents diffusion from one droplet to another and allows for the measurment of nucleation staisitic but also the thermodynamic of crystallization.
* Small angle X ray scattering. Small-Angle X-ray scattering (SAXS) is a powerful technique to investigate structures of soft matter and biological macromolecules at the nanometer-scale. It has been used for diverse applications, for example to study the nucleation of glycine crystals,colloidal silica, or to study proteins interactions in solution prior to crystallization, protein molecular weight, structure, or even conformational changes.
* Kinetic Monte Carlo Simulation : The efficiency of such numerical code is so good that one could manage systems up to a few picoliters by steps of nanometers, for milliseconds (continuous time), with medium-size workstations . These scales are close to what we can expect in microfluidic experiments, crediting this kind of simulations for applicability to real situations.

The combination of droplet based microfluidic and SAXS is a suitable tool to characterize all states of proteins during the crystallization process from form factor, structure factor to finally crystal form. This approach has been applied successfully to three different proteins, rasburicase, lysozyme and glucose isomerase. In the first experiment, the coupling between microfluidic set-up and SAXS was validated underlining the fact that the protein in droplets has the same behavior as in a usual solution. In addition, the best surfactant was chosen, tri-block copolymer type, which presented no radiation damage and no interaction with proteins within droplets.
In the next experiments, interactions between proteins in the presence of either salt or surfactant micelles were studied. Lysozyme was exposed to X-ray beam in presence of NaCl at various concentrations. The results show that without salt, the lysozyme solution is in repulsive regime and it changes to attractive regime when the salt concentration increases. By adding more salt, charges of lysozyme could be screened and the interaction between proteins becomes attractive.
In the last set of experiments, the scattering signals of crystals of glucose isomerase were recorded. Thanks to the presence of micelles formed by surfactant, the proteins could crystallize easily. When the concentration of surfactant within droplets was increased, higher and narrower signals were observed. This indicates that in droplets, more and bigger crystals of glucose isomerase were forming. Confirmation that the signal was from protein crystals was achieved by indexation of diffraction peaks.

Further work will focus on the integration with automated processing available at BM29 allowing feedback to guide the screening of buffer conditions, such as titrations for structural studies or to minimize interparticle scattering allowing acquisition from the best conditions. Further investigations currently planned are to understand the nucleation process of proteins, which is a crucial step in crystallization.
In conclusion, a versatile microfluidic tool which can be applied to numerous systems in a standardized way was developed. Using droplet microfluidic coupled with SAXS, structural studies of macromolecules in solution can be accomplished with significantly reduced sample quantity

Publication
1. N. V. Pham, D. Radajewski, P. Guillet, A. Round, M. Brennich, P. Pernot, B. Biscans, F. Bonnete´, S. Teychene´, Coupling digital microfluidics and Small-Angle X-ray scattering to study the whole crystallization process of proteins in solution Acta D Cryst 2015 (submitted)
International Conférences :
1. Coupling Droplet Microfluidic and Small Angle X-Ray Scattering N. V. Pham, D. Radajewski, P. Guillet, M. E. Brennich, A. Round, P. Pernot, B. Biscans, F. Bonneté, S. Teychené 19th International Symposium on Industrial Crystallization – 2014 - Toulouse France (ORAL)
2. Coupling droplet microfluidic and small angle X-ray scattering: Toward on-line measurement of first nucleation step , D. Radajewski, N. V. Pham, P. Guillet, M. E. Brennich, A. Round, P. Pernot, B. Biscans, F. Bonneté, S. Teychené 15th International Conference on the Crystallization of Biological Macromolecules – 2014 Hambourg- Germany (ORAL)
3. (INVITE) Coupling digital microfluidics and Small-Angle X-ray scattering to study the whole crystallization process of proteins in solution S. Teychene´, D. Radajewski, N. V. Pham, A. Round, M. Brennich, P. Pernot, B. Biscans, F. Bonnete´.
USER MEETING ESRF 2014 (ORAL)
4. Coupling digital microfluidics and Small-Angle X-ray scattering D. Radajewski, P. Guillet, M. E. Brennich, A. Round, P. Pernot, B. Biscans, F. Bonneté, S. Teychené, EMBL Conference: Microfluidics 2014 – Heidelberg (POSTER)
National Conferecens
1. (INVITE) Coupling digital microfluidics and Small-Angle X-ray scattering to study the whole crystallization process of proteins in solution S. Teychene´, D. Radajewski, N. V. Pham, A. Round, M. Brennich, P. Pernot, B. Biscans, F. Bonnete´.
Journée Scientifique C’nano Porquerolles 2014.

“Crystallization from solution must surely rank as the oldest unit operation, in the chemical engineering sense and it is known since the dawn of civilization” (Mullin, 1960). However, despite many experimental and theoretical developments, and despite its great interest in chemical, biological and natural processes, crystallization remains a puzzling phenomenon coupling several mechanisms (nucleation, growth, aggregation, agglomeration) occurring at different time and length scales. The crystallization process is still poorly understood mainly because crystal nucleation mechanisms are not clearly identified. The essential difficulties of studying nucleation and developing an accurate theory of the process arise from: 1/ the stochastic nature of nucleation, 2/ the fact that the objects involved during the process are small 3/ and are short lived (few ns).
In the CNOC project, we propose a basic research, combining original theoretical and experimental approaches involving a theoretical physicist, bio-physicists and chemical engineers, to get a better understanding of the nucleation mechanism involved during crystallization.
This project aims at a real breakthrough in our understanding of the nucleation process by associating modern experimental techniques (droplet microfluidics), powerful characterization technique (Small Angle X-Ray Scattering (SAXS) from a synchrotron source) and a new and original Monte Carlo simulation. The combination of microfluidics and SAXS enables in-situ measurements, at the nanoscale, of structures involved during the nucleation process and reduce the limit of time resolution at the micro/milli second range (due the spatial-to-time conversion offered by microfluidic devices). The theoretical description of nucleation from the Monte Carlo approach, enabling to treat complex and large systems (comparable to the one expected in microfluidics), will give a general picture of the mechanism involved during the nucleation.
The project stretches over 48 months, includes five main tasks and gathers researcher from four French academic laboratories internationally renowned: the Laboratoire de Génie Chimique in Toulouse, the Institut des Biomolécules Max Mousseron in Avignon, the Laboratoire de Physique des Solides in Orsay and the European Molecular Biology Laboratory in Grenoble providing complementary specialties, domains of knowledge, know-how and expertise for the good development of this inter- and multidisciplinary project.
Strong interactions between each participant will enable to design and build microfluidic chips compatible with intense X-ray radiations from a synchrotron source, to perform in-situ SAXS experiments for following molecular cluster in solution during the nucleation process, and to build a new theory of nucleation.
The results obtained, from a carefully chosen biological macromolecule model, in this project will highlight mechanisms involved during the nucleation process by following experimentally and numerically the temporal evolution of size, number and shape of the molecular clusters in solution: from the formation of pre-critical nuclei (before nucleation) until the emergence of the crystal.