(14b) Enhanced Microscale Flow and Controllable Phase Separation in an Evaporating Enzyme-Loaded Aqueous Two-Phase System (ATPS) Droplet

In living systems, biological and chemical reactions are commonly maintained away from thermodynamic equilibrium and display complex behaviors. To decipher the secrets of complicated but intelligent living systems step by step requires representative and controllable in vitro models. Besides, non-equilibrium structures can be modulated in unconventional manners when the system is out of equilibrium. Inspired by this, we establish a non-equilibrium model where enzymatic reaction and liquid-liquid phase separation (LLPS) can be coupled in droplets consisting of aqueous two-phase systems (ATPS).

We choose an ATPS consisting of polyethylene glycol (PEG) and dextran solutions and trigger phase separation by evaporation. Horseradish peroxidase conjugated antibody (HRP-dAb) is used to catalyze the Ampliflu Red(10-Acetyl-3,7-dihydroxyphenoxazine, ADHP). In the presence of HRP, the hydrogen peroxide (H₂O₂) can oxidize the non-fluorescent ADHP immediately into soluble, highly fluorescent Resorufin. When the HRP-ADHP catalysis is triggered in the PEG-dextran system, the reaction can be modulated due to affinity partitioning of ATPS. Specifically, the HRP-dAb preferentially diffuses into the dextran-rich phase while the ADHP and Resorufin molecules remain in the PEG-rich phase.

With the help of the microfluidic technique, the components of each enzyme-loaded ATPS droplet can be precisely tuned. The single-phase ATPS droplet is generated by the microfluidic device with two flow-focusing junctions. Before droplet generation, the substrate (ADHP and H₂O₂) is dissolved in the PEG-rich phase, and the enzyme HRP conjugated antibody is dissolved in the dextran-rich phase. Then the two aqueous phases meet and mix inside the microfluidic channel. Finally, the outermost phase of volatile HFE oil pinches the aqueous mixture into a single-phase droplet. After collecting the droplets, the phase separation of single-phase enzyme-loaded ATPS droplets is triggered by evaporation. We use FITC-dextran to label the dextran-rich phase and observe the non-equilibrium phenomena during the combined processes of LLPS and reaction.

Four groups of experiments are performed by varying the compositions of enzyme-loaded ATPS droplets. In Case One, the enzymatic reaction and phase separation happen simultaneously. The dextran phase gets concentrated, and the PEG sub-droplets emerge first during the evaporation process. Interestingly, the time for the droplet to vary from single-phase to two-phase droplets is shorter than the control group in Case four, which suggests that the enzymatic reaction may have sped up the phase separation. The internal flow inside the droplet is also enhanced, probably due to the oxygen bubbles generated by the catalysis reaction. In Case two, we do not involve the HRP-dAb, and no enzymatic reaction happens. The dextran is still concentrated during the phase separation. However, different from Case one, the dextran sub-droplets emerge first, unlike in Case One. One possible explanation for this is that the H₂O₂ depolymerizes the long dextran chains into shorter ones, thus transforming the dextran into the dispersed phase. In Case Three, we do not involve the substrate (ADHP, H₂O₂), and no enzymatic reaction takes place. As the dextran is concentrated, the emerge simultaneously. In Case Four, we have a control group consisting of PEG and dextran in buffer solutions. The PEG sub-droplets emerge first, and we did not observe a noticeable concentration of the dextran phase.

In summary, using the evaporating enzyme-loaded ATPS droplets, we have observed the non-equilibrium interfacial phenomena originated from the intertwined physics in enzymatic reaction and LLPS. The internal flow inside evaporating droplets can be enhanced by involving the reactants, and an accelerated LLPS is observed. Besides, the order in which the sub-droplets appear during the phase separation process can be controlled by tuning the compositions of the enzyme-loaded ATPS droplet. The varying dynamic states suggests that the enzymatic reaction can control the thermodynamic pathways of phase separation. The coupling between phase separation and reaction could have played an important role in biological systems, such as liquid organelles, and inspires new ways to control reactions via LLPS, and vice versa.

Acknowledgment：

This project is funded by the Research Grants Council of Hong Kong through the Collaborative Research Fund (No. C7165-20GF)