Crystallization of Proteins on Chip by Microdialysis for <i>In Situ</i> X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

Université Bordeaux

In this work, a viscosimeter implemented on a microfluidic chip is presented. The physical principle of this system is to use laminar parallel flows in a microfluidic channel. The fluid to be studied flows side by side with a reference fluid of known viscosity. By using optical microscopy, the shape of the interface between both fluids can be determined. Knowing the flow rates of the two liquids and the geometrical features of the channel, the mean shear rate sustained by the fluid and its viscosity can thus be computed. Accurate and precise measurements of the viscosity as a function of the shear rate can be made using less than 300 microL of fluid. Several complex fluids are tested with viscosities ranging from 10(-)(3) to 70 Pa.s.

Viscosimeter on a microfluidic chip.

Microfluidics offers a wide range of new tools that permit one to revisit the formation of crystals in solution and yield insights into crystallization processes. We review such recent microfluidic devices and particularly emphasize lab-on-chips dedicated to the high-throughput screening of crystallization conditions of proteins with nanolitre consumption. We also thoroughly discuss the possibilities offered by the microfluidic tools to acquire thermodynamic and kinetic data that may improve industrial processes and shed a new light on nucleation and growth mechanisms.

Microfluidic crystallization.

In most situations, drying is accompanied by the development of strong concentration gradients. Here, we evidence theoretically and experimentally that there exist microfluidic geometries for which confined drying becomes homogeneous, i.e., with no concentration gradient regardless of the type of solute involved in the process; ions, molecules, and colloids do concentrate in the same way providing a limited set of assumptions concerning the microfluidic geometry. It thus makes possible the establishment of phase diagrams of multi-component mixtures at the nanoliter scale. 

Drying with no concentration gradient in large microfluidic droplets.

We report an extensive study of the concentration process of aqueous polymer solutions confined within microfluidic channels, owing to the pervaporation of water through the matrix of the chip. Concentration of polymer continuously increases up to the formation of a dense material which eventually invades the channel. This technology can be used to fabricate micro-composites of different shapes starting from dilute inks. We use both theory and screening experiments to show that the dynamics of growth can be predicted by simple conservation equations. More precisely, we establish a quantitative prediction of the growth dynamics taking into account deformations of the soft channels of the moulds, and the solvent chemical activity of the polymer solution. The present results based on general transport equations for binary mixtures provide direct guidance for the design of micro-fabricated materials considering their shape, dimensions, time scale of fabrication, and chemical composition. This quantitative framework is indeed essential to engineer integrated polymer-based micro-devices by using combined pervaporation and microfluidic moulding. 

Investigation of the dynamics of growth of polymer materials obtained by combined pervaporation and micro-moulding.

We present an original microfluidic technique coupling pervaporation and the use of Quake valves to fabricate microscale materials (∼10 × 100 μm(2) × 1 cm) with composition gradients along their longest dimension. Our device exploits pervaporation of water through a thin poly(dimethylsiloxane) (PDMS) membrane to continuously pump solutions (or dispersions) contained in different reservoirs connected to a microfluidic channel. This pervaporation-induced flow concentrates solutes (or particles) at the tip of the channel up to the formation of a dense material. The latter invades the channel as it is constantly enriched by an incoming flux of solutes/particles. Upstream Quake valves are used to select which reservoir is connected to the pervaporation channel and thus which solution (or dispersion) enriches the material during its growth. The microfluidic configuration of the pervaporation process is used to impose controlled growth along the channel thus enabling one to program spatial composition gradients using appropriate actuations of the valves. We demonstrate the possibilities offered by our technique through the fabrication of dense assemblies of nanoparticles and polymer composites with programmed gradients of fluorescent dyes. We also address the key issue of the spatial resolution of our gradients and we show that well-defined spatial modulations down to ≈50 μm can be obtained within colloidal materials, whereas gradients within polymer materials are resolved on length scales down to ≈1 mm due to molecular diffusion. 

Fabrication of microscale materials with programmable composition gradients.

We investigate flow coating processes, i.e., the formation of dry coatings starting from dilute complex fluids confined between a static blade and a moving substrate. In particular, we focus on the evaporative regime encountered at low substrate velocity, at which the coating flow is driven mainly by solvent evaporation in the liquid meniscus. In this regime, general arguments based on mass conservation show that the thickness of the dry film decreases as the substrate velocity increases, unlike the behavior in the well-known Landau-Levich regime. This work focuses on colloidal dispersions, which deserve special attention. Indeed, flow coating is expected to draw first a solvent-saturated film of densely packed colloids, which further dries fully when air invades the pores of the solid film. We first develop a model based on the transport equations for binary mixtures, which can describe this phenomenon continuously, using appropriate boundary conditions and a criterion to take into account pore-emptying in the colloidal film. Extensive numerical simulations of the model then demonstrate two regimes for the deposit thickness as a function of the process parameters (substrate velocity, evaporation rate, bulk concentration, and particle size). We finally derive an analytical model based on simplified transport equations that can reproduce the output of our numerical simulations very well. This model can predict analytically the two observed asymptotic regimes and therefore unifies the models recently reported in the literature.

Modeling Flow Coating of Colloidal Dispersions in the Evaporative Regime: Prediction of Deposit Thickness.

In flow-coating processes at low substrate velocity, solvent evaporation occurs during the film withdrawal and the coating process directly yields a dry deposit. In this regime, often referred to as the evaporative regime, several works performed on blade-coating-like configurations have reported a deposit thickness h proportional to the inverse of the substrate velocity V. Such a scaling can be easily derived from simple mass conservation laws, assuming that evaporation occurs on a constant distance, referred to as the evaporation length, noted L in the present paper and of the order of the meniscus size. However, the case of colloidal dispersions deserves further attention. Indeed, the coating flow leads to a wet film of densely packed colloids before the formation of the dry deposit. This specific feature is related to the porous nature of the dry deposit, which can thus remain wet when capillary forces are strong enough to prevent the receding of the solvent through the pores of the film (the so-called pore-emptying). The length of this wet film may possibly be much larger than the meniscus size, therefore modifying the solvent evaporation rate, as well as the scaling h ∼ 1/V. This result was suggested recently by different groups using basic modeling and assuming for simplicity a uniform evaporation rate over the wet film. In this article, we go a step further and investigate the effect of multidimensional vapor mass transfer in the gas phase on L and h in the specific case of colloidal dispersions. Using simplified models, we first provide analytical expressions in asymptotic cases corresponding to 1D or 2D diffusive vapor transport. These theoretical investigations then led us to show that L is independent of the evaporation rate amplitude, and roughly independent of its spatial distribution. Conversely, h strongly depends on the characteristics of vapor mass transfer in the gas phase, and different scaling laws are obtained for the 1D or the 2D case. These theoretical findings are finally tested by comparison with experimental results supporting our theoretical simplified approach.

Role of Vapor Mass Transfer in Flow Coating of Colloidal Dispersions in the Evaporative Regime.

We report the fabrication of highly permeable membranes in poly(ethylene glycol) diacrylate (PEGDA) channels, for investigating ultra- or micro-filtration, at the microfluidic scale. More precisely, we used a maskless UV projection setup to photo-pattern PEG-based hydrogel membranes on a large scale (mm-cm), and with a spatial resolution of a few microns. We show that these membranes can withstand trans-membrane pressure drops of up to 7 bar without any leakage, thanks to the strong anchoring of the hydrogel to the channel walls. We also report in situ measurements of the Darcy permeability of these membranes, as a function of the deposited energy during photo-polymerization, and their formulation composition. We show that the use of PEG chains as porogens, as proposed in [Lee et al., Biomacromolecules, 2010, 11, 3316], significantly increases the porosity of the hydrogels, up to Darcy permeabilities of about 1.5 × 10-16 m2, while maintaining the strong mechanical stability of the membranes. We finally illustrate the opportunities offered by this technique, by investigating frontal filtration of colloidal dispersions in a straight microfluidic channel.

In situ photo-patterning of pressure-resistant hydrogel membranes with controlled permeabilities in PEGDA microfluidic channels.

We first report an original setup that enables continuous measurements of stresses induced by the drying of confined drops of complex fluids. This setup is mainly based on a precision scale working with an electromagnetic force compensation technique that provides accurate measurements of forces, while allowing simultaneously controlled evaporation rates, in situ microscopic observations, and thus quantitative estimates of normal stresses. We then performed an extensive study of the drying of a charged colloidal dispersion using this setup. Stress measurements clearly show the emergence of large tensile stresses during drying, well-before the solidification stage evidenced by the invasion of the porous colloidal material by air. Combined measurements of solid deformation and concentration profiles (particle tracking, Raman micro-spectroscopy) help us to demonstrate that these stresses are due to the formation of a solid at a low volume fraction, which further undergoes drying-induced shear deformations up to the colloid close-packing, as also supported by large deformation poroelastic modeling. Above all, our results highlight the importance of repulsive colloidal interactions in the build-up of mechanical stresses during drying.

Drying-induced stresses before solidification in colloidal dispersions: in situ measurements.

We report the fabrication of permeable membranes for microfluidic dialysis applications in poly(dimethylsiloxane) (PDMS) channels. A maskless UV projection device was used to photo-pattern long hydrogel membranes (mm-cm) with a spatial resolution of a few microns in PDMS chips integrating also micro-valves. We show in particular that multi-layer soft lithography allows one to deplete oxygen from the PDMS walls using a nitrogen gas flow and therefore makes possible in situ UV-induced polymerization of hydrogels. We also report a simple surface modification of the PDMS channels leading to strongly anchored hydrogel membranes that can withstand trans-membrane pressure drops up to 1 bar without leakages. We then measured the Darcy permeability of these membranes and estimated their cut-off by measuring the kinetics of diffusion of macromolecules of different sizes through the membrane. Finally, we illustrate the opportunities offered by such microfluidic chips for dialysis applications by observing in real time the crystallization of a model protein in a chamber of a few nanoliters.

Microfluidic dialysis using photo-patterned hydrogel membranes in PDMS chips.

In the present work, we use Mach-Zehnder interferometry to thoroughly investigate the drying dynamics of a 2D confined drop of a charged colloidal dispersion. This technique makes it possible to measure the colloid concentration field during the drying of the drop at a high accuracy (about 0.5%) and with a high temporal and spatial resolution (about 1 frame per s and 5 μm per pixel). These features allow us to probe mass transport of the charged dispersion in this out-of-equilibrium situation. In particular, our experiments provide the evidence that mass transport within the drop can be described by a purely diffusive process for some range of parameters for which the buoyancy-driven convection is negligible. We are then able to extract from these experiments the collective diffusion coefficient of the dispersion D(φ) over a wide concentration range φ = 0.24-0.5, i.e. from the liquid dispersed state to the solid glass regime, with a high accuracy. The measured values of D(φ) ≃ 5-12D0 are significantly larger than the simple estimate D0 given by the Stokes-Einstein relation, thus highlighting the important role played by the colloidal interactions in such dispersions.

Jean-Baptiste Salmon

CNRS,
Solvay LOF,
UMR 5258,
CNRS, Solvay LOF, UMR 5258

Université Bordeaux

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

Jean-Baptiste Salmon

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}CNRS,Solvay LOF,UMR 5258,CNRS, Solvay LOF, UMR 5258

Université Bordeaux

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

CNRS,
Solvay LOF,
UMR 5258,
CNRS, Solvay LOF, UMR 5258