Thermo-responsive aqueous two-phase system for two-level compartmentalization

Abstract

Hierarchical compartmentalization responding to changes in intracellular and extracellular environments is ubiquitous in living eukaryotic cells but remains a formidable task in synthetic systems. Here we report a two-level compartmentalization approach based on a thermo-responsive aqueous two-phase system (TR-ATPS) comprising poly(N-isopropylacrylamide) (PNIPAM) and dextran (DEX). Liquid membraneless compartments enriched in PNIPAM are phase-separated from the continuous DEX solution via liquid-liquid phase separation at 25 °C and shrink dramatically with small second-level compartments generated at the interface, resembling the structure of colloidosome, by increasing the temperature to 35 °C. The TR-ATPS can store biomolecules, program the spatial distribution of enzymes, and accelerate the overall biochemical reaction efficiency by nearly 7-fold. The TR-ATPS inspires on-demand, stimulus-triggered spatiotemporal enrichment of biomolecules via two-level compartmentalization, creating opportunities in synthetic biology and biochemical engineering.

Fig. 1. Schematic of two-level compartmentalization of TR-ATPS.

a Schematics of two-level compartmentalization of the PNIPAM/DEX system. b Biomolecular storage and (c) enzyme cascade reaction in the colloidosome-like compartments.

Fig. 2. Compartmentalization of the PNIPAM/DEX system at different temperatures.

Phase-separated PNIPAM droplets in PNIPAM (5 wt%)/DEX (5 wt%) solution under (a) FITC channel, (b) rhodamine channel, and (c) merged channel at 25 °C. Confocal images of PNIPAM droplets in PNIPAM (5 wt%)/DEX (5 wt%) solution under (d) FITC channel, (e) rhodamine channel, and (f) merged channel at 25 °C. g Histogram illustrating the size distribution of primary compartments at 25 °C. Results are collected from more than n  >  20 different primary compartments. h The fluorescence intensity profile shows the distribution of PNIPAM and DEX along the white dash line in (f). Phase-separated PNIPAM droplets in PNIPAM (5 wt%)/DEX (5 wt%) solution under (i) FITC channel, (j) rhodamine channel, and (k) merged channel at 35 °C. Confocal images of PNIPAM droplets in PNIPAM (5 wt%)/DEX (5 wt%) solution under (l) FITC channel, (m) rhodamine channel, and (n) merged channel at 35 °C. o Histogram illustrating the size distribution of primary compartments at 35 °C. Results are collected from more than n  >  20 different primary compartments. p The fluorescence intensity profile shows the distribution of PNIPAM and DEX along the white dash line in (n). Confocal images show the colloidosome-like surface structure under (q) FITC channel, (r) rhodamine channel, and (s) merged channel. The cross-section of the hierarchical multi-compartment under (t) FITC channel, (u) rhodamine channel, and (v) merged channel. Z-stack confocal images of colloidosome-like compartments formed in the PNIPAM (5 wt%)/DEX (5 wt%) system under (w) merged channel.

Fig. 3. Thermally reversible two-level compartmentalization of the PNIPAM/DEX system at different temperatures.

a Schematic illustration of generating PNIPAM/DEX all-aqueous double emulsions by droplet microfluidics. b Schematic diagram displaying the phase transition in the resulting all-aqueous double emulsions. All-aqueous double emulsions with dex-rich cores and PNIPAM-rich shells under (c) FITC channel, (d) rhodamine channel, and (e) merged channel at 25 °C. Each experiment was repeated independently three times with similar results. f Fluorescence intensity profile shows the distribution of PNIPAM and DEX along with the white dash line in (e). The transition of all-aqueous double emulsions to condensed shells with diluted cores under (g) FITC channel, (h) rhodamine channel, and (i) merged channel at 35 °C. Each experiment was repeated independently three times with similar results. j Fluorescence intensity profile shows the distribution of PNIPAM and DEX along with the white dash line in (i).

Fig. 4. Mechanism of two-level compartmentalization.

a Schematic of two-level compartmentalization. b ATR-FTIR spectra of the PNIPAM (5 wt%)/DEX (5 wt%) aqueous solution at 25 °C and 35 °C. c ¹H-NMR spectroscopy of the 5 wt% PNIPAM, 5 wt% DEX, and PNIPAM (5 wt%)/DEX (5 wt%) aqueous solution at 25 and 35 °C. d Phase-separated PNIPAM droplets in PNIPAM (5 wt%)/PAM (5 wt%) solution under FITC channel, rhodamine channel, and merged channel at 25 and 35 °C. Each experiment was repeated independently three times with similar results. e Phase-separated PEG droplets in PEG (1 wt%)/DEX (9 wt%) solution under FITC channel, rhodamine channel, and merged channel at 25 and 35 °C. Each experiment was repeated independently three times with similar results.

Fig. 5. Biomolecular storage in TR-ATPS.

a Schematic of biomolecules storage in TR-ATPS. Storage and release of (b) FAM-RNA in PNIPAM (5 wt%)/DEX (5 wt%) aqueous solution at 35 and 25 °C. Each experiment was repeated independently three times with similar results.

Fig. 6. Spatiotemporal regulation of bi-enzymatic cascade reactions through compartmentalization of GOX and HRP in PNIPAM/DEX systems.

a Schematic diagram and chemical reaction equations demonstrating the generation of resorufin catalyzed by GOX and HRP. Colocalization of GOX and HRP in the small second-level compartments from the PNIPAM (5 wt%)/DEX (5 wt%) under (b) rhodamine channel, (c) FITC channel, and (d) merged channel at 35 °C. The confocal images show the colocalization of GOX and HRP on the (e) surface and (f) cross-section of the colloidosome-like compartments under the merged channel at 35 °C. g Partitioning fraction of GOX in PNIPAM-rich phase from PNIPAM/DEX systems at 25 °C and 35 °C. h Partitioning fraction of HRP in DEX-rich phase from PNIPAM/DEX systems at 25 °C and 35 °C. i Partitioning fraction of HRP in PNIPAM-rich phase from PNIPAM/DEX systems at 25 °C and 35 °C. Michaelis–Menten plots of tandem reactions catalyzed by GOX and HRP in (j) a 50 mM sodium phosphate buffer (pH 7.4), (k) a 50 mM sodium phosphate buffer (pH 7.4) containing PEG (1 wt%)/DEX (9 wt%), and (l) a 50 mM sodium phosphate buffer (pH 7.4) containing PNIPAM (5 wt%)/DEX (5 wt%) at 25 and 35 °C. Lineweaver–Burk plots of tandem reactions catalyzed by GOX and HRP in (m) a 50 mM sodium phosphate buffer (pH 7.4), (n) a 50 mM sodium phosphate buffer (pH 7.4) containing PEG (1 wt%)/DEX (9 wt%), and (o) a 50 mM sodium phosphate buffer (pH 7.4) containing PNIPAM (5 wt%)/DEX (5 wt%) at 25 and 35 °C. Error bars indicate mean ± SD (n  =  3 independent samples).