A Double Emulsion-Based, Plastic-Glass Hybrid Microfluidic Platform for Protein Crystallization

Deyong Zhu,Xiaohu Zhou,Bo Zheng

doi:10.3390/mi6111446

Abstract

This paper reports the design and construction of a plastic-glass hybrid microfluidic platform for performing protein crystallization trials in nanoliter double emulsions. The double emulsion-based protein crystallization trials were implemented with both the vapor-diffusion method and microbatch method by controlling the diffusion of water between the inner and outer phases and by eliminating water evaporation. Double emulsions, whose inner and outer environments can be easily adjusted, can provide ideal conditions to explore protein crystallization with the advantages of a convection-free environment and a homogeneous interface. The property of the water-oil interface was demonstrated to be a critical factor for nucleation, and appropriate surfactants should be chosen to prevent protein adsorption at the interface. The results from the volume effect study showed a trend of fewer crystals and longer incubation time when the protein solution volume became smaller, suggesting that the nucleation in protein crystallization process can be controlled by changing the volume of protein solutions. Finally, sparse matrix screening was achieved using the double emulsion-based microbatch method. The double emulsion-based approach for protein crystallization is a promising tool for enhancing the crystal quality by controlling the nucleation process.

Highlights

The atomic structure of protein molecules is important both for understanding the architectures and functions of living matter and for providing essential knowledge to the pharmaceutical industry, such as new drug discovery and medical therapy [1,2,3]
There have been several techniques developed for determining the three-dimensional protein structures in laboratories, including X-ray crystallography [4,5,6,7], nuclear magnetic resonance spectroscopy (NMR) [8] and cryo-electron microscopy [9,10]
We developed a new plastic-glass hybrid microfluidic platform for producing and stabilizing nanoliter-scale double emulsions and demonstrated the application of double emulsions for protein crystallization

Summary

Introduction

The atomic structure of protein molecules is important both for understanding the architectures and functions of living matter and for providing essential knowledge to the pharmaceutical industry, such as new drug discovery and medical therapy [1,2,3]. Chayen et al [36] developed a “containerless” crystallization technique by suspending a droplet of crystallizing reagents between two immiscible kinds of oils, one of which has a higher density than water and the other one lower, to make the droplet float at the interface Compared to those protein crystallization trials performed in single emulsions, double emulsions can provide ideal conditions to explore protein crystallization for the advantages of a convection-free environment and a homogeneous interface. During all steps, heating the silicone tubing for a few minutes can soften the tubing and make the insertion of Teflon tubing or glass capillaries significantly easier This hybrid microfluidic device needs no surface treatment, as Teflon in the first junction is non-wettable to all of the reagents used in this work and suitable for producing water-in-oil emulsions, while the glass in the second junction is hydrophilic enough for oil-in-water emulsion generation. Replacing or washing each of the two junctions is easy for the purpose of reuse

Microfluidic Production of Emulsions

Reagents and Data Analysis for Protein Crystallization

Findings

Conclusions