Reversible Light-Responsive Coacervate Microdroplets With Rapid Regulation Of Enzymatic Reactions Rate

Membrane-free coacervate microdroplets are widely utilized as protocell or membrane-less organelle models in the study of cell physicochemical properties.Based on the close connection between the assembly/disassembly of condensates in vivo and pathology,constructing coacervate microdroplets in vitro and realizing flexible dynamic regulation are of great significance for exploring the abilities of condensates to response and promote biological functions.For this reason,this article proposes a membrane-free protocell model based on light-responsive coacervate microdroplets.Compared with ion control,p H,and temperature control,light control has favourable spectral and sensitive spatiotemporal capabilities,which will provide more precise regulation for the simulation of biological functions related to condensates in vivo.Firstly,the anion succinyl amylose(Su-Amy)and the cation azobenzene trimethylammonium bromide(azo TAB)with photoisomerization properties are combined by liquid-liquid phase separation(LLPS)to form light-responsive coacervate microdroplets.Coacervate microdroplets quickly disassemble within 10 seconds under ultraviolet light(UV,λ=365 nm,405 nm),and reassemble after irradiated with blue light(λ=460 nm,488nm)in about 630 seconds.The light-controlled disassembly-reassembly process can be performed at least 7 times,and the coacervate size is stable in the range of1.98±0.37μm to 2.43±0.41μm.The formation mechanism of coacervate microdroplets and the mechanism of light-controlled behavior are explored.The electrostatic shielding of Na Cl and the hydrophobic destruction of urea confirmed that electrostatic and hydrophobic interactions are the dual driving forces for liquid-liquid phase separation.According to ~1H-NMR calculations,azo TAB has a planar trans-configuration(50.43%)under blue light,and a curved cis-configuration(94.65%)under UV light.By measuring the UV-vis spectra of the azo TAB solution before and after mixing Su-Amy,it is proved that Su-Amy is mainly combined with the trans-configuration of azo TAB.The electrostatic attraction of the positive and negative groups of the two components and the insertion of hydrophobic ends of trans-azo TAB with the hydrophobic domains on the Su-Amy helical structure promotes the light-controlled behavior of the coacervate microdroplets.A series of guest molecules with different properties are selected to test the sequestration ability of coacervate microdroplets.Based on the strong electrostatic and hydrophobic binding forces inside the microdroplets,more hydrophobic or oppositely charged guest molecules such as Nile red and Peroxidase from horseradish(HRP)can be sequestrated to increase their local concentrations,while hydrophilic and negatively charged molecules are isolated from the outside.Taking advantage of this characteristic,combined with the rapid and accurate response of the microdroplets to external light,we propose a method to flexibly and reversibly regulate the enzymatic reaction rates by the light-induced assembly-disassembly-reassembly of coacervate microdroplets.The enzymatic reaction under blue light reaches the end within 6 minutes,while it takes longer under UV light.Therefore,we realize the regulation of the enzymatic reaction rate by changing the external light(blue or UV light).