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New COC Microfluidic Devices for In-Situ Diffusion-Based Macromolecular Crystallography Show Better X-Ray Compatibility

The study of structural biology has been rapidly advancing due to the availability of new technology. The malleable nature of microfluidics has made it a primary candidate for analyzing biological forms.

Study: Manufacturing of Ultra-Thin X-ray-Compatible COC Microfluidic Devices for Optimal In Situ Macromolecular Crystallography Experiments. Image Credit: Explode/Shutterstock.com

The cyclic-olefin-copolymer (COC)-based microfluidic approach has emerged as a frontrunner in this endeavor.  The main objective of a recently undertaken project by Professor Leonard Chavas’s group at Synchrotron Radiation Research Center, Nagoya University, was to create COC microfluidic apparatuses for in situ diffraction investigations at hard X-ray synchrotron sources, as well as diffusion-based macromolecular crystallography. The research findings were published in MDPI-Micromachines.

What is Microfluidics?

Microfluidics is used to investigate the behavior of fluids through micro-channels.  Scientific and technological development is carried out to create miniaturized devices that can handle fluid flow constrained within narrow pathways.

On a micrometric scale, fluids exhibit different properties than under regular conditions. Fluid quantities as little as femtoliters can be transported in microfluidic channels. These distinctive characteristics are crucial for new scientific research and inventions.

Cyclic-Olefin-Copolymer (COC)-Based Microfluidic Devices

Device fabrication for microfluidics has employed various materials and methods. Some of these are:

  • Polydimethylsiloxane
  • Norland Optical Adhesive
  • OrmoComp
  • Hot embossing
  • Cyclic olefin copolymers (COCs)
  • Laser structuring (Kapton)

COC has emerged as the material for microfluidic applications due to its exceptional qualities and high purity level.

Some of the challenges faced in traditional fabrication methods for microfluidic devices lie in their limitations in fast prototyping.

COC lends itself to quick prototyping and is compatible for X-ray scattering, which is a widely used technique for characterizing structural biology.

X-ray scattering is particularly useful in crystallography. The experimental exploration for determining crystals' atomic and molecular structure is known as crystallography. In-depth knowledge of such structures aids in understanding the properties of crystals better and builds better tools in mineralogy and chemistry.

COC is a straightforward material for manufacturing microfluidic channels thanks to its thermoplastic properties.

X-ray Diffraction Crystallography

The X-ray diffraction studies used in macromolecular crystallography include measuring the intensities of light diffracted by a sample subjected to an X-ray beam.

These intensities are challenging to detect and measure because of the background noise produced by diffusing materials that the incoming X-ray beam passes through. The microfluidic devices must be modified in this situation and to improve MX experiments to prevent adding unneeded noise to the experiments. This is challenging as most of the device materials used to create the devices absorb X-rays.

Professor Chavas’s group set out to fabricate COC-based microfluidic devices that would reduce the background noise on in-situ X-ray diffraction experiments.  Macromolecular crystallography to analyze diffusion-based crystallization is also another goal of the project.

Experimental Details

A step-wise fabrication method was used to produce the COC device. First, UV-lithography was employed to produce a photoresist master. Soft lithography was applied to prepare a Polydimethylsiloxane (PDMS) stamp from the master. The PDMS stamp was later used to hot-emboss the final perfluoroalkoxy (PFA) polymer.

A detailed description of the experimental process from photo-lithographic master fabrication, COC device fabrication, sample loading and crystallization to data acquisition, processing, and structure determination is given in the MDPI-Micromachines article.

One of the challenges faced in manufacturing diffusion-based crystallization devices is in making microscopic features from large amounts of polymer. Due to the conducive features of COCs, such as weak adhesion to substrates and its natural elasticity, they successfully maintained stable bonds. This led to COC devices that could be used for Macromolecular crystallography for diffusion-based crystallization.

The research group grew Lysozyme crystals in the newly constructed COC microfluidic channels. An experimental technique adapted in this device was to use a separate inlet and outlet at the ends of the chip.

Lysozyme crystals were confined within the channel width between the chip ends. This novel design mitigates the background noise associated with X-ray scattering usually encountered in such chip architectures. The results showed much less radiation damage from X-ray radiation on the materials. Further data analysis also confirmed improved crystallographic statistics and better quality microfluidics.

The Future of Microfluidics

The results obtained by Professor Chavas and colleagues highlight the potential and versatility of microfluidics. COC-based devices achieve better sensitivity in X-ray diffraction tests by mitigating background noise when used in conjunction with synchrotron radiation sources.

Expanding on the technique by adding more inlets and tube channels, diffusion properties can be examined in more detail. Specifically targeted drug tests can be incorporated with COC and microfluidic devices in the future.

References

Vasireddi, Ramakrishna, Antonin Gardais, and Leonard M. G. Chavas. (2022) Manufacturing of Ultra-Thin X-ray-Compatible COC Microfluidic Devices for Optimal In Situ Macromolecular Crystallography Experiments. Micromachines, 13, no. 8: 1365. https://www.mdpi.com/2072-666X/13/8/1365/htm

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Written by

Ilamaran Sivarajah

Ilamaran Sivarajah is an experimental atomic/molecular/optical physicist by training who works at the interface of quantum technology and business development.

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