In situ counter-diffusion crystallization and long-term crystal preservation in microfluidic fixed targets for serial crystallography

Compared with batch and vapor diffusion methods, counter diffusion can generate larger and higher-quality protein crystals yielding improved diffraction data and higher-resolution structures. Typically, counter-diffusion experiments are conducted in elongated chambers, such as glass capillaries, and the crystals are either directly measured in the capillary or extracted and mounted at the X-ray beamline. Despite the advantages of counter-diffusion protein crystallization, there are few fixed-target devices that utilize counter diffusion for crystallization. In this article, different designs of user-friendly counter-diffusion chambers are presented which can be used to grow large protein crystals in a 2D polymer microfluidic fixed-target chip. Methods for rapid chip fabrication using commercially available thin-film materials such as Mylar, propyl-ene and Kapton are also detailed. Rules of thumb are provided to tune the nucleation and crystal growth to meet users' needs while minimizing sample consumption. These designs provide a reliable approach to forming large crystals and maintaining their hydration for weeks and even months. This allows ample time to grow, select and preserve the best crystal batches before X-ray beam time. Importantly, the fixed-target microfluidic chip has a low background scatter and can be directly used at beamlines without any crystal handling, enabling crystal quality to be preserved. The approach is demonstrated with serial diffraction of photoactive yellow protein, yielding 1.32 Å resolution at room temperature. Fabrication of this standard microfluidic chip with commercially available thin films greatly simplifies fabrication and provides enhanced stability under vacuum. These advances will further broaden microfluidic fixed-target utilization by crystallographers

A Miniaturized Programmable Multi-fluidic pneumatic for precise controls of sample preparation environment

High-density microfluidics is becoming an important experimental platform for studying complex biological systems such as synthetic gene regulatory networks, molecular biocomputating of engineered cells, distributing rapid point-of-care diagnosis, and monitoring pathological environment. Imaging transient bio-chemical reactions happening in these systems at a single particle or cellular level requires precise time-dependent control of sample reaction and imaging conditions at the desired fluidic momentum. In this study, we showed our novel miniaturized and programmable electronic-based pneumatic system to meet the requirement. We demonstrated its capability to control reaction parameters such as concentrations and injection rates in a liposome production system

Advanced manufacturing provides tailor-made solutions for crystallography with x-ray free-electron lasers.

Serial crystallography at large facilities, such as x-ray free-electron lasers and synchrotrons, evolved as a powerful method for the high-resolution structural investigation of proteins that are critical for human health, thus advancing drug discovery and novel therapies. However, a critical barrier to successful serial crystallography experiments lies in the efficient handling of the protein microcrystals and solutions at microscales. Microfluidics are the obvious approach for any high-throughput, nano-to-microliter sample handling, that also requires design flexibility and rapid prototyping to deal with the variable shapes, sizes, and density of crystals. Here, we discuss recent advances in polymer 3D printing for microfluidics-based serial crystallography research and present a demonstration of emerging, large-scale, nano-3D printing approaches leading into the future of 3D sample environment and delivery device fabrication from liquid jet gas-dynamic virtual nozzles devices to fixed-target sample environment technology

Advanced manufacturing provides tailor-made solutions for crystallography with x-ray free-electron lasers

Serial crystallography at large facilities, such as x-ray free-electron lasers and synchrotrons, evolved as a powerful method for the high-resolution structural investigation of proteins that are critical for human health, thus advancing drug discovery and novel therapies. However, a critical barrier to successful serial crystallography experiments lies in the efficient handling of the protein microcrystals and solutions at microscales. Microfluidics are the obvious approach for any high-throughput, nano-to-microliter sample handling, that also requires design flexibility and rapid prototyping to deal with the variable shapes, sizes, and density of crystals. Here, we discuss recent advances in polymer 3D printing for microfluidics-based serial crystallography research and present a demonstration of emerging, large-scale, nano-3D printing approaches leading into the future of 3D sample environment and delivery device fabrication from liquid jet gas-dynamic virtual nozzles devices to fixed-target sample environment technology

AN OPEN-CHANEL MICROFLUIDIC MEMBRANE DEVICE FOR IN SITU HYPERSPECTRAL MAPPING OF ENZYMATIC CELLULOSE HYDROLYSIS

Synchrotron infrared hyperspectral microscopy is a label-free and non-invasive technique well suited for imaging of chemical events in situ. It can track the spatial and temporal distributions of molecules of interests in a specimen in its native state by the molecule’s characteristic vibrational modes. Despite tremendous progress made in recent years, IR hyperspectral imaging of chemical events in biomaterials in liquids remains challenging because of the demanding requirements on environmental control and strong infrared absorption of water. Here we report a multi-phase capillary-driven membrane device for label-free and real-time investigation of enzymatic deconstruction of algal cellulose purified from Cladophora aegagropila

All polymer microfluidic chips-A fixed target sample delivery workhorse for serial crystallography.

The development of x-ray free electron laser (XFEL) light sources and serial crystallography methodologies has led to a revolution in protein crystallography, enabling the determination of previously unobtainable protein structures and near-atomic resolution of otherwise poorly diffracting protein crystals. However, to utilize XFEL sources efficiently demands the continuous, rapid delivery of a large number of difficult-to-handle microcrystals to the x-ray beam. A recently developed fixed-target system, in which crystals of interest are enclosed within a sample holder, which is rastered through the x-ray beam, is discussed in detail in this Perspective. The fixed target is easy to use, maintains sample hydration, and can be readily modified to allow a broad range of sample types and different beamline requirements. Recent innovations demonstrate the potential of such microfluidic-based fixed targets to be an all-around workhorse for serial crystallography measurements. This Perspective will summarize recent advancements in microfluidic fixed targets for serial crystallography, examine needs for future development, and guide users in designing, choosing, and utilizing a fixed-target sample delivery device for their system

Development and Characterization of Bio-Based Composite Films for Food Packing Applications Using Boiled Rice Water and <i>Pistacia vera</i> Shells

Customer demand for natural packaging materials in the food industry has increased. Biocomposite films developed using boiled rice water could be an eco-friendly and cost-effective packaging product in the future. This study reports the development of bio-based films using waste materials, such as boiled rice water (matrix) and Pistacia vera shells (reinforcement material), using an adapted solution casting method. Several film combinations were developed using various concentrations of plasticizing agent (sorbitol), thickening agent (oil and agar), and stabilizing agents (Arabic gum, corn starch, and Pistacia vera shell powder). Various packaging properties of the film were analyzed and examined to select the best bio-based film for food packaging applications. The film fabricated with Pistacia vera shell powder in the biocomposite film exhibited a reduced water solubility, swelling index, and moisture content, as compared to polyethene packaging material, whereas the biocomposite film exhibited poor antimicrobial properties, high vapor transmission rate, and high biodegradability rate. The packaging properties and characterization of the film indicated that the boiled rice water film with Pistacia vera shell powder was suitable for packaging material applications

A user-friendly plug-and-play cyclic olefin copolymer-based microfluidic chip for room-temperature, fixed-target serial crystallography.

Over the past two decades, serial X-ray crystallography has enabled the structure determination of a wide range of proteins. With the advent of X-ray free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and structure determination. A crucial need to continue advancement is the efficient delivery of fragile and micrometre-sized crystals to the X-ray beam intersection. This paper presents an improved design of an all-polymer microfluidic `chip for room-temperature fixed-target serial crystallography that can be tailored to broadly meet the needs of users at either synchrotron or XFEL light sources. The chips are designed to be customized around different types of crystals and offer users a friendly, quick, convenient, ultra-low-cost and robust sample-delivery platform. Compared with the previous iteration of the chip [Gilbile et al. (2021), Lab Chip, 21, 4831-4845], the new design eliminates cleanroom fabrication. It has a larger imaging area to volume, while maintaining crystal hydration stability for both in situ crystallization or direct crystal slurry loading. Crystals of two model proteins, lysozyme and thaumatin, were used to validate the effectiveness of the design at both synchrotron (lysozyme and thaumatin) and XFEL (lysozyme only) facilities, yielding complete data sets with resolutions of 1.42, 1.48 and 1.70 Å, respectively. Overall, the improved chip design, ease of fabrication and high modifiability create a powerful, all-around sample-delivery tool that structural biologists can quickly adopt, especially in cases of limited sample volume and small, fragile crystals