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Study Of New Techniques For High-throughput Protein Crystallization Screening Based On Microfluidic Systems

Posted on:2017-05-03Degree:MasterType:Thesis
Country:ChinaCandidate:Y R LiangFull Text:PDF
GTID:2311330512468790Subject:Chemistry
Abstract/Summary:PDF Full Text Request
Currently, X-ray crystallography is the most important method for structure identification of proteins, which requires diffraction-quality crystals. However, obtaining protein crystals mainly relies on large-scale trial screening of a tremendous chemical space with a high consumption of protein. Microfluidic technology offers reliable methods for protein crystallization screening, taking advantages of high-throughput, low consumption, integration and promotion of crystallization.In the 1st chapter, the methodology of protein crystallization and screening is described and commercial platforms designed for protein crystallization are introduced. We also review the methods and techniques for protein crystallization and screening based on microfluidic technology. In addition, we introduce the application of three dimentional (3D) printing technology in fabrication of microfluidic chips.In the 2nd chapter, we describe the implementation of nanoliter-scale vapor diffusion-based protein crystallization and screening using a 3D-printed microfluidic chip and droplet technology.3D-printing technique makes it possible for the fabrication of crystallization chambers including wells with high-aspect-ratio and different volumes. To accomplish a long-term incubation for protein crystallization in these chambers, we developed a two-step post processing for 3D-printed chip and a multi-layer structure, contributing to fully-sealed chambers and inert chamber surface respectively. A microfluidic droplet robot was employed for assembling of nanoliter-volume droplet reactors in a fully-automated way. Efficient and reliable vapor diffusion experiment with nanoliter droplets was confirmed by hydrating and dehydrating with lower and higher concentration salt solutions. The dehydration speed was optimized and adjusted to facilitate crystal growth. We applied the established system for crystallization screening of two model proteins with commercial precipitants. The protein consumption of each trial in this system is 10-100 times less than that of conventional protein crystallization.In the 3 rd chapter, we developed a new method for protein crystallization and screening named High Resolution Microbatch (HRMB). In HRMB, precipitant droplets were dispensed in the micro-well array in the microfluidic chip firstly. Then, the controllable concentration gradient of protein solution was formed in a capillary, and a droplet-array with different concentrations was dispensed on a microfluidic chip with the second generation of the sequential operation droplet array system. These droplets are mixed with precipitant droplets. We used this system for crystallization and screening of 5 model proteins under 480 commercial screening conditions. Every condition included 12 trails with different protein concentration, consuming only 40 nL of protein solution.
Keywords/Search Tags:microfluidics, protein crystallization, 3D printing, concentration gradient, high-throuput screening
PDF Full Text Request
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