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Microfluidic Platforms for Advanced Crystallography

X-ray crystallography has played a critical role in structural biology, allowing for the direct study of the interplay between protein structure and function. However, the fragile nature of protein crystals creates significant challenges with respect to crystal handling, including the potential for physical damage and crystal dehydration. Microfluidic and microscale technologies have the potential to facilitate protein crystallization and structure determination in a protected environment, as well as high-throughput in situ data collection, without the need to manipulate crystals after growth. Furthermore, X-ray compatible microfluidics can enable advanced structural studies that would be otherwise inaccessible if more traditional crystal mounting strategies were employed. Our goal is to establish innovative microfluidic platforms as next-generation sample delivery strategies for X-ray crystallography. We developed a novel microfluidic device architecture that takes advantage of large-area sheets of graphene to facilitate X-ray crystallographic analysis of protein crystals. The use of atomically-thin graphene films minimizes the amount of material surrounding a crystal while serving as a vapor-diffusion barrier that is stable against significant water loss over the course of weeks. This approach enables long-term incubation of protein crystallization trials and direct in situ analysis of the resulting crystals. Building on this capability, we demonstrated the potential for using this device architecture to enable the collection of high quality X-ray diffraction data under the presence of an electric field, and have applied this platform to enable new protein crystallization techniques, such as electro-crystallization. We have also used microfluidic technology to address limitations in the high-throughput workflow associated with screening of potential drug targets. We used an innovative fabrication strategy to develop photoresist-based microfluidic array devices to accelerate compound screening efforts in early-stage drug discovery We demonstrated a new compound screening workflow based on our microfluidic platform with significantly improved efficiency in both sample preparation and data collection. We validated our devices by obtaining high-resolution ligand-protein complex structures from protein crystals soaked with ligands in various conditions. This work paves way for the adoption of microfluidics as a strategy for advanced sample delivery at synchrotron X-ray sources, particularly for large-scale pharmaceutical endeavors
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