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

Abstract

This paper reports a composite microfluidic system for performing protein crystallization trials in nanoliter aqueous droplets inside capillaries suitable for X-ray diffraction and the evaluation of the quality of the crystals directly by on-chip X-ray diffraction. Crystallization conditions can be screened with both microbatch and vapor-diffusion techniques by eliminating evaporation of solutions and by controlling diffusion of water between droplets.Growing of high-quality crystals of proteins and other macromolecules plays an important role in structural biology. Crystallization conditions are usually identified by performing a large number of trials in which variable ratios of solutions of a protein, precipitants, and additives are pipetted together by hand (≈1 μL droplets) or with a robotic dispenser (≈100-10 nL droplets). [1] This process could be improved by a system that minimizes the amount of protein sample consumed, reduces labor, and is simple and economical enough to be implemented in individual laboratories. Micro-fluidics could serve as a platform for such a system because it allows sophisticated handling of small volumes (nL-pL) of reagents in a potentially simple, inexpensive format. [2] In addition, microfluidics may provide an opportunity to perform experiments that are difficult to do on larger scale-for example, Hansen et al. have crystallized proteins on a polydimethylsiloxane (PDMS) microfluidic device by free interface diffusion. [3] Previously, we screened crystallization conditions [4] on a PDMS microfluidic chip by using nanoliter droplets [5] formed in microfluidic channels.In this work, we rely on the same droplet-based platform, but take three steps that advance this work beyond our previous report. 1) We implemented the microbatch technique, in which we completely eliminated problems of uncontrolled evaporation through PDMS. PDMS is waterand oil-permeable, and the crystallization of proteins in PDMS, at least in our hands,

Controlling Nonspecific Protein Adsorption in a Plug-Based Microfluidic System by Controlling Interfacial Chemistry Using Fluorous-Phase Surfactants

“…Therefore, k a obtained from the initial rate of adsorption should adequately describe the process. The rate of adsorption that accounts for time-dependent protein concentration C t can be expressed as dθ t / C t = k a dt (6) By using θ t = Γ(t)/Γ max and eq 5 to express θ t in terms of C t , the integrated form of eq 6 is ln (C t / C 0 ) = − k a t(SA / V )Γ max C t = C 0 exp ( − k a t(SA / V )Γ max ) (7) To relate adsorption in droplets and in plugs, we calculated C t as a function of time using eq 7. To predict how adsorption in plugs would affect enzyme kinetics, we used C t to numerically solve for product formation according to Michaelis-Menten kinetics.…”

Multi-step synthesis of nanoparticles performed on millisecond time scale in a microfluidic droplet-based system

“…19 We have used this droplet-based microfluidic method to perform rapid kinetics measurements of single stage reactions 20 and to perform protein crystallizations. 21,22 Several methods of forming droplets in microfluidic channels have been described, and gas bubbles have also been used to enhance the mixing of liquids. 1,[23][24][25][26] To perform multi-step, time controlled syntheses with this droplet-based microfluidic method, additional reagent must be added to the droplets formed from initial reagents.…”

Encapsulation of single cells into monodisperse droplets by fluorescence-activated droplet formation on a microfluidic chip

“…The compartmentalization of reactions in droplets provides rapid mixing of reagents, flexible control of sample volumes, and prevention of water evaporation and cross contamination between samples [1][2][3]. These advantages of droplet-based microfluidics have shown benefits to a wide range of chemical and biological applications, such as enzymatic kinetics [4][5][6], protein crystallization [7][8][9], polymerase chain reaction [10][11][12] and clinical diagnosis [13].…”