CrystalChip - Microfluidic chips for protein crystallization

The counter-diffusion method consists in directly mixing the sample and precipitant solutions by putting them in contact in a restricted geometry. As soon as the sample and precipitant solution are loaded, a continuous gradient of concentrations is created by liquid-liquid diffusion. This system allows to explore a wide range of supersaturation conditions in a single experiment while keeping sample volumes to a minimum.

Although the counter-diffusion method has already proven its worth as an excellent choice for screening purposes, its widespread adoption has been so far restricted by experimental setup complexity, limited access to sophisticated equipments and costly microfabrication techniques. 

CrystalChip makes counter-diffusion directly available to researchers by offering a user-friendly & standardized screening tool. In addition, its microfluidics design implemented on a standard microscope slide format unlocks potential to add ligands, directly visualize crystal growth, determine crystal structure in situ without handling and store high-quality crystals on the long-term, opening exciting perspectives in crystallography! 

No, we do not recommend using CrystalChip in cryogenic conditions. As multiple microcrystals will form along each channel, the CrystalChip design is fully optimized for serial crystallization collection at room temperature, which has the advantage of operating at more physiological temperatures and does not require any handling of the crystals. If using a XFEL, each crystal introduced into the beam will produce a single image before being destroyed. If using a synchrotron with a beam less intense, a partial dataset is usually collected on each crystal before radiation damage, thus reducing the number of samples required to determine a structure.

Unlike traditional data collection performed on single crystal rotating in the X-ray beam at cryogenic temperature, a series of randomly oriented crystals is analyzed at room temperature to reconstruct a complete diffraction space. Serial crystallography has the advantage of operating at more physiological temperatures and allows crystals to reveal their full diffraction potential after characterized by a very low mosaicity. Serial crystallography also unlocks real-time monitoring of dynamic processes within crystals, such as enzymatic catalysis, opening a new field of investigation in structural biology with the possibility of producing real molecular films. 

Precise control and reproducibility of crystallization conditions are essential in serial crystallography, as the process depends on producing multiple crystals of consistent size and quality, often with limited biomolecule samples. The CrystalChip’s microfluidic design supports extreme miniaturization and high-throughput serial crystallization experiments, offering a convection-free environment that promotes the growth of high-quality crystals. Additionally, it provides an efficient means of positioning crystals in the X-ray beam for serial diffraction experiments.

CrystalChip has been successfully used to crystallize various soluble & membrane proteins, alone or in complex with substrates. Successful crystals have been obtained with the following biomolecules (non-exhaustive list): 
- CCA-adding enzyme 
- lysozyme
- thaumatin
- insulin
- nanobody from llama
- protease
- lipase
- tRNA synthetases (human & bacterial)
- membrane transporters
- hemoglobin
- RNA duplex

Find out example results and more detailed information on buffer, crystallant solutions used & resolutions achieved for each protein in the Results & Publications sections.

Total duration for crystal formation can vary greatly depending on each individual biomolecule characteristics. Some proteins will crystallize in a few minutes (i.e. lysozyme) up to a few days or weeks. Chips are typically checked under the microscope over a period of 2-4 weeks to track the growth of crystals.

CrystalChip is a user-friendly tool that does not require any sophisticated equipment. We provide you with microfluidic chips, accurate pipet tips for sample loading and a holder to position your chip on synchrotron or XFEL facilities. In option, you can add ready-to-use crystallization solutions to your order. All you need to have on your end is your purified protein and, if needed, some paraffin oil. 

We strongly recommend using the provided crystallization solutions when using CrystalChip for initial screening of conditions. These crystallization cocktails, made of the most successful precipitating agents typically employed in protein crystallization, were specifically optimized for the counter-diffusion method and were shown to crystallize all samples in a previous study testing 14 different proteins [1]. 

Alternatively, you can load your own precipitating solution and use CrystalChip as an optimization tool, treat your crystals with ligands or analyse them in situ. 

We do not recommend using standard commercial kits for initial screening with CrystalChip. The counter-diffusion method will work best with precipitating agents concentrated up to the maximum of their solubility at 4°C, which is not typically the case with commercial kits designed for vapour diffusion or batch methods. If initial crystallization conditions were obtained by vapor diffusion, the crystallant concentration should be increased by a factor of 1.5-2.  

[1] Efficient Screening Methodology for Protein Crystallization Based on the Counter-Diffusion Technique. Luis A. González-Ramírez, Carlos R. Ruiz-Martínez, Rafael A. Estremera-Andújar, Carlos A. Nieves-Marrero, Alfonso García-Caballero, José A. Gavira, Juan López-Garriga, and Juan M. García-Ruiz. Crystal Growth & Design 2017 17 (12), 6780-6786. DOI: 10.1021/acs.cgd.7b01353

You can label your protein with a fluorescent compound to facilitate crystal identification and their discrimination from salt crystals.

Crystals cannot be extracted from their microfluidic channels once formed in CrystalChip. Yet, the CrystalChip method provides a fully integrated pipeline for the determination of crystal structure, in which all steps are carried out in situ at room temperature. Therefore, there is no need to retrieve the crystals or perform the diffraction analysis at cryogenic conditions. This avoids altering crystal mosaicity by physical interaction or cryocooling, preserving their genuine diffraction properties. 

All our CrystalChip kits include a 3D-printed chip holder, that was specifically designed to attach electromagnets present on standard goniometers. This holder has been notably used for X-ray analysis at diverse synchrotron facilities across Europe (SOLEIL & ESRF, France and SLS, Switzerland). 

Alternatively, it is also possible to load the chip in a universal frame (i.e. microplate format) if a robotic arm is used, or if using the chip in an imager. Chip dimensions are that of a standard microscope slide (25mmx75mm), so it can be placed in any commercially available frame that will fit that format. Please contact us at [email protected] for further advice. 

The CrystalChip has been used for X-ray analysis at diverse synchrotron facilities across Europe, and diffraction data were collected on the following beamlines:

•  Swiss Light Source (SLS), Villigen, Switzerland: beamline PXII equipped with a PILATUS 6M detector & beamline PXIII equipped with a MAR CCD or a PILATUS 2M-F detector, X10SA and X06DA beamlines equipped with a MAR MX225 CCD detector or DECTRIS PILATUS 2M/6M pixel detectors.
• SOLEIL, Saint-Aubin, France: PROXIMA-2A (PX2A) beamline equipped with an EIGER X 9M detector
• ESRF, Grenoble, France: beamline ID30B equipped with a PILATUS3 6M detector, FIP-BM30A beamline equipped with an ADSC Quantum 315r CCD detector.

Once entry ports are sealed, the microfluidic chip provides a solid and air-tight system in which crystals can be safely transported and stored for several months while preserving their intrinsic quality. Some protein crystals were used after 3 years !

Yes, CrystalChip can be loaded in an imager system for direct crystal visualization.  The CrystalChip has dimensions of a standard microscope slide (25mm x 75mm), and will fit on any compatible insert. Please contact us at [email protected] if you need further advice.

CrystalChip has been specifically designed to prepare crystals for analysis by X-ray diffraction and has not been directly applied to cryo-EM so far. Indeed, once crystals have formed in the microfluidic chip, they are typically kept inside the chip for in situ analyses as it can be quite tricky to fish them out. Analyses that can be carried out directly in the chip include X-ray crystallography, serial crystallography and also direct optical imaging methods (fluorescence imaging, polarized light, etc) as the chip is at a standard microscope format. That being said, CrystalChip can provide a useful complementary tool to use alongside single particle cryo-EM to quickly generate X-ray crystallographic atomic models alongside cryo-EM workflows. This can be particularly useful if you want to get a more complete structural picture of a molecule, dock atomic models into a cryo-EM map or resolve small or highly dynamic structures that may present challenges for cryo-EM. Indeed, performing X-ray analyses in the chip requires very little protein solution, and the protocol is really straight-forward (you simply have to inject your protein & crystallization solutions, and all analyses are performed in situ at room temperature).  

Lipidic cubic phases are unfortunately too viscous to be injected inside the microfluidic system of the chips. That being said, CrystalChip can very well be used for membrane protein crystallization. Membrane protein crystals have been successfully obtained in the chip using membrane protein solutions containing a detergent (cf paper below). 

2019 | A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography
de Wijn R, Hennig O, Roche J, Engilberge S, Rollet K, Fernandez-Millan P, Brillet K, Betat H, Mörl M, Roussel A, Girard E, Mueller-Dieckmann C, Fox GC, Olieric V, Gavira JA, Lorber B, Sauter C. IUCrJ. 2019 Apr 19;6(Pt 3):454-464. doi: 10.1107/S2052252519003622. PMID: 31098026; PMCID: PMC6503916.

These microfluidic chips take advantage of the counter-diffusion principle. As soon as the sample and precipitant solution are loaded, a continuous gradient of concentrations is created by liquid-liquid diffusion: each substance has a higher concentration at the end where it was introduced, creating strong, opposing gradients that drive diffusion. This is made possible by the geometry of the capillary channels, providing a convection-free environment that is prone to maintaining well-defined concentration gradients for both substances (the flow is dominated by diffusion rather than convection because of the small dimensions of the channels).

Crystals formed in the CrystalChip are micron-sized, in accordance with the 80µm x 80µm channels. 

A few dozens of crystals are typically required to determine a structure using CrystalChip when using synchrotron beamlines. Unlike XFELs, synchrotron beams are less intense so it is generally possible to measure a partial dataset on each crystal by collecting small rotation wedges before radiation damage causes the resolution to drop.

Although these chips have been initially developed for biomolecule crystallization, they could be very useful for resolving the structures of other types of molecules too. Relying on the counter-diffusion principle, they provide a very convenient tool to maximize your chance of finding the right conditions for crystallization while saving reagents. They can also offer a well-suited environment to flow external compounds (i.e. ligands, hit molecules, etc) on a crystallized compound.

The main things to be careful about when crystallizing small molecules in the chip is the choice of the solvent, which has to be compatible with the chip material. The chip is made of a copolymer (COC), and will therefore be compatible with most organic polar solvents. Non-polar/halogenated solvents such as chloroform or dichloromethane should be avoided as they will alter the material. Dimethylformamide (DMF) has already been successfully used with the CrystalChip. 

Another important parameter to take into account is the choice of the crystallization solutions. Indeed, those provided in the "screening kits" were optimized to screen for biomolecule crystallization conditions and contain a series of concentrated PEG & ammonium sulphate solutions with pH ranging from 3 to 10. Precise composition and concentration of those used for your molecule might need to be experimentally determined.

The chip is made of a copolymer (COC), and will therefore be compatible with most organic polar solvents. Non-polar/halogenated solvents such as chloroform or dichloromethane should be avoided as they will alter the material. Dimethylformamide (DMF) has already been successfully used with the CrystalChip. 

Here is a table detailing the chip compatibility with different various solvents: