Fabrication of X-ray compatible microfluidic platforms for protein crystallization

Abstract

This paper reports a method for fabricating multilayer microfluidic protein crystallization platforms using different materials to achieve X-ray transparency and compatibility with crystallization reagents. To validate this approach, three soluble proteins, lysozyme, thaumatin, and ribonuclease A were crystallized on-chip, followed by on-chip diffraction data collection. We also report a chip with an array of wells for screening different conditions that consume a minimal amount of protein solution as compared to traditional screening methods. A large number of high quality isomorphous protein crystals can be grown in the wells, after which slices of X-ray data can be collected from many crystals still residing within the wells. Complete protein structures can be obtained by merging these slices of data followed by further processing with crystallography software. This approach of using an x-ray transparent chip for screening, crystal growth, and X-ray data collection enables room temperature data collection from many crystals mounted in parallel, which thus eliminates crystal handling and minimizes radiation damage to the crystals.

Microfluidics: From crystallization to serial time-resolved crystallography

“…The subsequent development of vacuum actuate-to-open valves enabled the design of array-style counter-diffusion chips that could be set up and then allowed to incubate without the need for ancillary pressure sources (Figures 1(d) and 1(e)). 18,19,39,40 Furthermore, this new generation of devices was designed specifically with in situ X-ray analysis in mind. These chips retained only the thin layer of PDMS necessary for valve actuation, replacing the remaining device layers with thin films of cyclic olefin copolymers (COCs) to alleviate the potential for sample dehydration during incubation and/or data collection (Figure 1(d)).…”

“…These issues are then further compounded by the need to deliver those samples efficiently to the X-ray beam. 5,[7][8][9][10][11]18,[37][38][39][40][41][42] Microfluidic and microscale technologies have played a critical role in facilitating both protein crystallization and structure determination, with the breadth and variety of reported solutions demonstrating the challenging nature of the field. In this review, we discuss the utility of microfluidic technologies for addressing the challenges of crystal growth and sample manipulation for applications in serial crystallography and time-resolved structure determination.…”

Can Microfluidics boost the Map of Glycome Code?

“…The analysis on the single cell keeps the 3D organization of the glycans and the cross linked structure, as consequence the analysis by MS gives only limitative results losing the information on the space structure of the glycans, the conformation that could promote the crosstalk with other organisms. The standard techniques to perform 3D analysis of the cells can be done by NMR and X-ray techniques that also can exploit the advantage of miniaturization, even if the available examples are at the moment negligible [81,82]. …”

A Graphene-Based Microfluidic Platform for Electrocrystallization and In Situ X-ray Diffraction

“…Such platforms have been increasingly harnessed to facilitate the diffraction analysis of challenging targets for both static and dynamic structure determination. Various platforms have been developed to improve the growth and subsequent mounting of tiny and fragile crystals for X-ray diffraction analysis [24][25][26][27][28], including dense array-style devices [29][30][31][32][33][34][35][36][37][38][39][40][41], platforms for the lipidic cubic phase crystallization of membrane proteins [42,43], and thin-film sandwich devices [44]. In the meantime, the challenges of such platforms lie in the need to maintain a protected sample environment, as well as minimize the interference of device materials with the subsequent X-ray analysis.…”