(20f) Microfluidic Platforms for In Situ Crystallographic Analysis

Protein crystallography is an important aspect of structural biology, which provides insight into protein structure as well as further information about their function. There are many steps in the process which finally leads to the structure of the protein and subsequently unravels how it functions. These involve expression, purification, crystallization, harvesting of the crystals for diffraction and finally X-ray data analysis. Growing diffraction quality crystals and manually harvesting them still remains a major challenge. The difficulty in obtaining quality crystals stems from the fact that crystallization conditions for novel proteins are not known a priori. Traditionally, sparse matrix screens need to be set up to obtain suitable crystallization conditions and this requires large amounts of protein and is a labor intensive process. Recent developments in robotics have allowed researchers to set up thousands of trials in a short duration of time (1). However, the ability to set up a large number of trials does not overcome challenges associated with the reproducibility of crystallization events or crystal quality.  Furthermore, these efforts are limited by the need to manually harvest fragile crystals.

Microfluidics offers exquisite control over transport phenomena, which provides precise control over composition and kinetics of a crystallization experiment than what is possible at the macroscale (2). However, to take advantage of these benefits, it is necessary to couple complex fluid handling capabilities with the ability to perform on-chip crystallographic analysis.

The multilayer microfluidic crystallization platforms described here fabricated out of X-ray transparent materials which allow in situ analysis and can be used for high throughput experiments. Our 96-well screening and optimization chips can screen eight different protein-to-precipitant ratios for twelve different precipitant conditions using less than 2µL of protein. A suitable condition once identified, can be scaled out in an array format to reproducibly grow a large number of crystals. 

We are currently working on extending the present chip architecture, which has been validated for in situ screening, to perform time resolved studies on protein dynamics. Laue crystallography is a technique which is used to perform time resolved studies on protein dynamics and we have validated our chip architecture for Laue diffraction. Here we aim to couple in situ analysis of a large number of crystals with the capabilities of a microfluidic flow cell to enable the controlled introduction of various chemical stimuli and thus trigger biologically relevant conformational changes in the structure which can then be analyzed in situ in our microfluidic chips.

 References:

1.     Stevens RC (2000) High-throughput protein crystallization. Current Opinion in Structural Biology 10(5):558-563.

2.     Zheng B, Tice JD, Roach LS, & Ismagilov RF (2004) A droplet-based, composite PDMS/glass capillary microfluidic system for evaluating protein crystallization conditions by microbatch and vapor-diffusion methods with on-chip X-ray diffraction. Angewandte Chemie-International Edition 43(19):2508-2511.