Rapid Determination of Phase Diagrams for Biomolecular Liquid–Liquid Phase Separation with Microfluidics

Abstract

Biomolecular phase separation is currently emerging in both the medical and life science fields. Meanwhile, the application of liquid–liquid phase separation has been extended to many fields including drug discovery, fibrous material fabrication, 3D printing, and polymer design. Although more than 8600 proteins and other synthetic macromolecules are capable of phase separation as recently reported, there is still a lack of a high-throughput approach to quantitatively characterize its phase behaviors. To meet this requirement, here, we proposed fast and high-resolution acquisition of biomolecular phase diagrams using microfluidic chips. Using this platform, we demonstrated the phase behavior of polyU/RRASLRRASLRRASL in a quantitative manner. Up to 1750 concentration conditions can be generated in 140 min. The detection limitation of our device to capture the saturation concentration for phase separation is about 5 times lower than that of the traditional turbidity method. Thus, our results provide a basis for the rapid acquisition of phase diagrams with high-throughput and pave the way for its wide application.

Microfluidics for multiscale studies of biomolecular condensates

“…An alternative approach has been reported, which loaded and mixed small volumes of materials on a centrifugal chip and determine the phase boundary by sampling through a gradient of concentrations (Figure 3D) [62]. Protein and another solution were injected to a disc-like microfluidic device, which contain 25 reaction units.…”

“…(E) (i) A centrifugal microfluidic chip for phase diagrams, each containing 25 units (ii) when the sample was loaded, the difference in metering channel lengths leads to varied mixing ratio. Centrifugation forced the solutions into the reaction chamber to mix 62. (F) A design for droplet-based screening of rational crystallisation.…”

Droplet Microfluidics for the Label‐Free Extraction of Complete Phase Diagrams and Kinetics of Liquid–Liquid Phase Separation in Finite Volumes

“…[14] Typically, the cloud-point temperature (T cp ) is evaluated by varying temperatures at one fixed polymer concentration or, alternatively, the saturation concentration is estimated by progressively increasing the polymer concentration at constant temperature (Figure 1A). Recent techniques based on droplet-based microfluidics [15][16][17][18][19][20][21] and Taylor dispersion analysis [22] enable these measurements to be performed at high-throughput. These methods have been successfully applied to evaluate the left arm of the binodal, which is the coexistence curve that defines the two-phase region (Figure 1A).…”

Automated Centrifugal Microfluidic Chip Integrating Pretreatment and Molecular Diagnosis for Hepatitis B Virus Genotyping from Whole Blood

“…Unlike other microfluidic devices, the centrifugal microfluidic chip can be driven only by rotation rather than extra bulky syringe pumps and cumbersome tubing systems. Using physical forces including centrifugal force, the Coriolis force, and the Euler force on centrifugal platforms, fluids can be controlled precisely to realize various functions such as transporting, valving, metering, aliquoting, mixing, and so on, − which provides a great potential for integrating a variety of complicated functions into a sample-in-answer-out platform for POC molecular diagnosis. Furthermore, the centrifugal microfluidic chip can also incorporate adjustable valves easily for new exploration, such as thermopneumatic valves, laser-actuated valves, wax valves, and so on.…”