Spatial Positioning and Chemical Coupling in Coacervate‐in‐Proteinosome Protocells

The integration of molecularly crowded microenvironments into membrane‐enclosed protocell models represents a step towards more realistic representations of cellular structure and organization. Herein, the membrane diffusion‐mediated nucleation of either negatively or positively charged coacervate microdroplets within the aqueous lumen of individual proteinosomes is used to prepare nested hybrid protocells with spatially organized and chemically coupled enzyme activities. The location and reconfiguration of the entrapped droplets are regulated by tuning the electrostatic interactions between the encapsulated coacervate and surrounding negatively charged proteinosome membrane. As a consequence, alternative modes of a cascade reaction involving membrane‐ and coacervate‐segregated enzymes can be implemented within the coacervate‐in‐proteinosome protocells.     

Self-programmed enzyme phase separation and multiphase coacervate droplet organization

Membraneless organelles are phase-separated droplets that are dynamically assembled and dissolved in response to biochemical reactions in cells. Complex coacervate droplets produced by associative liquid–liquid phase separation offer a promising approach to mimic such dynamic compartmentalization. Here, we present a model for membraneless organelles based on enzyme/polyelectrolyte complex coacervates able to induce their own condensation and dissolution. We show that glucose oxidase forms coacervate droplets with a cationic polysaccharide on a narrow pH range, so that enzyme-driven monotonic pH changes regulate the emergence, growth, decay and dissolution of the droplets depending on the substrate concentration. Significantly, we demonstrate that time-programmed coacervate assembly and dissolution can be achieved in a single-enzyme system. We further exploit this self-driven enzyme phase separation to produce multiphase droplets via dynamic polyion self-sorting in the presence of a secondary coacervate phase. Taken together, our results open perspectives for the realization of programmable synthetic membraneless organelles based on self-regulated enzyme/polyelectrolyte complex coacervation.

Self-programmed enzyme phase separation is exploited to assemble dynamic multiphase coacervate droplets via spontaneous polyion self-sorting under non-equilibrium conditions.     

Catanionic Coacervate Droplets as a Surfactant‐Based Membrane‐Free Protocell Model

We report on the formation of surfactant‐based complex catanionic coacervate droplets in mixtures of decanoic acid and cetylpyridinium chloride or cetyltrimethylammonium bromide. We show that coacervation occurs over a broad range of composition, pH, and ionic strength. The catanionic coacervates consist of elongated micelles, sequester a wide range of solutes including water‐soluble organic dyes, polysaccharides, proteins, enzymes, and DNA, and can be structurally stabilized by sodium alginate or gelatin‐based hydrogelation. These results suggest that catanionic coacervates could be exploited as a novel surfactant‐based membrane‐free protocell model.     

Construction of coacervate-in-coacervate multi-compartment protocells for spatial organization of enzymatic reactions

Coacervate microdroplets, formed via liquid–liquid phase separation, have been extensively explored as a compartment model for the construction of artificial cells or organelles. In this study, coacervate-in-coacervate multi-compartment protocells were constructed using four polyelectrolytes, in which carboxymethyl-dextran and diethylaminoethyl-dextran were deposited on the surface of as-prepared polydiallyldimethyl ammonium/deoxyribonucleic acid coacervate microdroplets through layer-by-layer assembly. The resulting multi-compartment protocells were composed from two immiscible coacervate phases with distinct physical and chemical properties. Molecule transport experiments indicated that small molecules could diffuse between two coacervate phases and that macromolecular enzymes could be retained. Furthermore, a competitive cascade enzymatic reaction of glucose oxidase/horseradish peroxidase–catalase was performed in the multi-compartment protocells. The different enzyme organization and productions of H₂O₂ led to a distinct polymerization of dopamine. The spatial organization of different enzymes in immiscible coacervate phases, the distinct reaction fluxes between coacervate phases, and the enzymatic cascade network led to distinguishable signal generation and product outputs. The development of this multi-compartment structure could pave the way toward the spatial organization of multi-enzyme cascades and provide new ideas for the design of organelle-containing artificial cells.

A coacervate-in-coacervate micro-architecture is constructed as a multi-compartment protocell model, in which a multi-enzyme cascade is spatially organized for competitive enzymatic reactions.     

Modulation of Higher‐order Behaviour in Model Protocell Communities by Artificial Phagocytosis

Collective behaviour in mixed populations of synthetic protocells is an unexplored area of bottom‐up synthetic biology. The dynamics of a model protocell community is exploited to modulate the function and higher‐order behaviour of mixed populations of bioinorganic protocells in response to a process of artificial phagocytosis. Enzyme‐loaded silica colloidosomes are spontaneously engulfed by magnetic Pickering emulsion (MPE) droplets containing complementary enzyme substrates to initiate a range of processes within the host/guest protocells. Specifically, catalase, lipase, or alkaline phosphatase‐filled colloidosomes are used to trigger phagocytosis‐induced buoyancy, membrane reconstruction, or hydrogelation, respectively, within the MPE droplets. The results highlight the potential for exploiting surface‐contact interactions between different membrane‐bounded droplets to transfer and co‐locate discrete chemical packages (artificial organelles) in communities of synthetic protocells.     

Multistimuli responsive ternary polyampholytes: Formation and crosslinking of coacervates

A series of multiresponsive ternary polyampholytes were prepared by free‐radical copolymerization of N‐(3‐aminopropyl) methacrylamide hydrochloride (APM), methacrylic acid (MAA), and N‐(2‐hydroxyethyl) acrylamide (HEA). APM and MAA were held at 1:1 molar ratio, while the HEA monomer feed was varied between 14 and 33 mol %. Compositional drift during polymerization was monitored by ¹H nuclear magnetic resonance, and minimized by adjusting the reactivity of MAA through its degree of ionization. The resulting polyampholytes phase‐separate from aqueous solution to form coacervate droplets, depending on HEA content, pH, ionic strength, and temperature. These coacervate droplets could be covalently crosslinked and the resulting hydrogel particles were found to swell with increasing ionic strength. Such soluble and microgel polyampholytes open opportunities for new multistimuli responsive biomaterials. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 2109–2118     

Microfluidic Formation of Membrane‐Free Aqueous Coacervate Droplets in Water

We report on the formation of coacervate droplets from poly(diallyldimethylammonium chloride) with either adenosine triphosphate or carboxymethyl‐dextran using a microfluidic flow‐focusing system. The formed droplets exhibit improved stability and narrower size distributions for both coacervate compositions when compared to the conventional vortex dispersion techniques. We also demonstrate the use of two parallel flow‐focusing channels for the simultaneous formation and co‐location of two distinct populations of coacervate droplets containing different DNA oligonucleotides, and that the populations can coexist in close proximity up to 48 h without detectable exchange of genetic information. Our results show that the observed improvements in droplet stability and size distribution may be scaled with ease. In addition, the ability to encapsulate different materials into coacervate droplets using a microfluidic channel structure allows for their use as cell‐mimicking compartments.     

Reversible pH‐Responsive Coacervate Formation in Lipid Vesicles Activates Dormant Enzymatic Reactions

In situ, reversible coacervate formation within lipid vesicles represents a key step in the development of responsive synthetic cellular models. Herein, we exploit the pH responsiveness of a polycation above and below its pK_a, to drive liquid–liquid phase separation, to form single coacervate droplets within lipid vesicles. The process is completely reversible as coacervate droplets can be disassembled by increasing the pH above the pK_a. We further show that pH‐triggered coacervation in the presence of low concentrations of enzymes activates dormant enzyme reactions by increasing the local concentration within the coacervate droplets and changing the local environment around the enzyme. In conclusion, this work establishes a tunable, pH responsive, enzymatically active multi‐compartment synthetic cell. The system is readily transferred into microfluidics, making it a robust model for addressing general questions in biology, such as the role of phase separation and its effect on enzymatic reactions using a bottom‐up synthetic biology approach.     

Hierarchical Proteinosomes for Programmed Release of Multiple Components

A facile route to hierarchically organized multicompartmentalized proteinosomes based on a recursive Pickering emulsion procedure using amphiphilic protein–polymer nanoconjugate building blocks is described. The number of incarcerated guest proteinosomes within a single host proteinosome is controlled, and enzymes and genetic polymers encapsulated within targeted subcompartments to produce chemically organized multi‐tiered structures. Three types of spatiotemporal response—retarded concomitant release, synchronous release or hierarchical release of dextran and DNA—are demonstrated based on the sequential response of the host and guest membranes to attack by protease, or through variations in the positioning of disulfide‐containing cross‐links in either the host or guest proteinosomes integrated into the nested architectures. Overall, our studies provide a step towards the construction of hierarchically structured synthetic protocells with chemically and spatially integrated proto‐organelles.     

Catch and Release of DNA in Coacervate‐Dispersed Gels

Summary: We have synthesized novel coacervate‐droplet gels, which were applied to controlling the transportation of DNA in electrophoresis. Coacervate droplets are colloidal particles and they are usually composed of positive and negative polyelectrolytes. However, the polyzwitterion (polyampholyte) PDMAPS can form coacervate droplets in water by itself, since PDMAPS has both positive and negative charges in each side group of main chain. Coacervate droplets have a unique nature and can catch charged macromolecules such as DNA. In order to utilize the nature of the PDMAPS coacervate droplets for the catch and release of DNA, we stabilized PDMAPS droplets in gels. The droplets catch the DNA in electrophoresis and the release of DNA can be controlled by temperature and salt addition.

Electrophoresis of DNA, using coacervate‐dispersed PAAm gels, prepared at various PDMAPS concentrations. The circles indicate the PAAm gels embedded in agarose gels.     

Fuel-driven macromolecular coacervation in complex coacervate core micelles

Fuel-driven macromolecular coacervation is an entry into the transient formation of highly charged, responsive material phases. In this work, we used a chemical reaction network (CRN) to drive the coacervation of macromolecular species readily produced using radical polymerisation methods. The CRN enables transient quaternization of tertiary amine substrates, driven by the conversion of electron deficient allyl acetates and thiol or amine nucleophiles. By incorporating tertiary amine functionality into block copolymers, we demonstrate chemical triggered complex coacervate core micelle (C3M) assembly and disassembly. In contrast to most dynamic coacervate systems, this CRN operates at constant physiological pH without the need for complex biomolecules. By varying the allyl acetate fuel, deactivating nucleophile and reagent ratios, we achieved both sequential signal-induced C3M (dis)assembly, as well as transient non-equilibrium (dis)assembly. We expect that timed and signal-responsive control over coacervate phase formation at physiological pH will find application in nucleic acid delivery, nano reactors and protocell research.

We apply an allyl acetate fuelled chemical reaction network (CRN) to control the coacervation of macromolecular species at constant physiological pH without the need for complex biomolecules.     

Autonomic Behaviors in Lipase‐Active Oil Droplets

Developing self‐fueled micro‐reactor droplets with programmable autonomic behaviors provides a step towards smart liquid dispersions comprising motile microscale objects. Herein, we prepare aqueous suspensions of lipase‐coated oil globules comprising a mixture of a triglyceride substrate (tributyrin, 1,2,3‐tributylglycerol) and a low‐density oil (polydimethylsiloxane, PDMS) and describe a range of active behaviors based on controlled enzyme‐mediated consumption of individual droplets under non‐equilibrium conditions. Encapsulation of the lipase‐coated lipid/PDMS droplets into a model protocell as energy‐rich sub‐compartments is demonstrated as an internalized mechanism for activating protocell buoyancy. Taken together, our results highlight opportunities for the regulation of autonomic behavior in enzyme‐powered oil droplets and provide a new platform for increasing the functionality and energization of synthetic protocells.     

Study on Glucose Biofuel Cells Using an Electrochemical Noise Device

An electrochemical noise (ECN) device was utilized for the first time to study and characterize a glucose/O₂ membraneless biofuel cell (BFC) and a monopolar glucose BFC. In the glucose/O₂ membraneless BFC, ferrocene (Fc) and glucose oxidase (GOD) were immobilized on a multiwalled carbon nanotubes (MWCNTs)/Au electrode with a gelatin film at the anode; and laccase (Lac) and an electron mediator, 2,2′‐azinobis (3‐ethylbenzothiazoline‐6‐sulfonate) diammonium salt (ABTS), were immobilized on a MWCNTs/Au electrode with polypyrrole at the cathode. This BFC was performed in a stirred acetate buffer solution (pH 5.0) containing 40 mmol/L glucose in air, with a maximum power density of 8 μW/cm², an open‐circuit cell voltage of 0.29 V, and a short‐circuit current density of 85 μA/cm², respectively. The cell current at the load of 100 kΩ retained 78.9% of the initial value after continuous discharging for 15 h in a stirred acetate buffer solution (pH 5.0) containing 40 mmol/L glucose in air. The performance decrease of the BFC resulted mainly from the leakage of the ABTS mediator immobilized at the cathode, as revealed by the two‐channel quartz crystal microbalance technique. In addition, a monopolar glucose BFC was performed with the same anode as that in the glucose/O₂ membraneless BFC in a stirred phosphate buffer solution (pH 7.0) containing 40 mmol/L glucose, and a carbon cathode in Nafion‐membrane‐isolated acidic KMnO₄, with a maximum power density of 115 μW/cm², an open‐circuit cell voltage of 1.24 V, and a short‐circuit current density of 202 μA/cm², respectively, which are superior to those of the glucose/O₂ membraneless BFC. A modification of the anode with MWCNTs for the monopolar glucose BFC increased the maximum power density by a factor of 1.8. The ECN device is highly recommended as a convenient, real‐time and sensitive technique for BFC studies.     

DNA-Based Artificial Receptors as Transmembrane Signal Transduction Systems for Protocellular Communication

The ability to reproduce signal transduction and cellular communication in artificial cell systems is significant in synthetic protobiology. Here, we describe an artificial transmembrane signal transduction through low pH-mediated formation of the i-motif and dimerization of DNA-based artificial membrane receptors, which is coupled to the occurrence of fluorescence resonance energy transfer and the activation of G-quadruplex/hemin-mediated fluorescence amplification inside giant unilamellar vesicles. Moreover, an intercellular signal communication model is established when the extravesicular H⁺ input is replaced by coacervate microdroplets, which activate the dimerization of the artificial receptors, and subsequent fluorescence production or polymerization in giant unilamellar vesicles. This study represents a crucial step towards designing artificial signalling systems with environmental response, and provides an opportunity to establish signalling networks in protocell colonies.     

Reversible pH-Responsive Coacervate Formation in Lipid Vesicles Activates Dormant Enzymatic Reactions

In situ, reversible coacervate formation within lipid vesicles represents a key step in the development of responsive synthetic cellular models. Herein, we exploit the pH responsiveness of a polycation above and below its pK_a, to drive liquid–liquid phase separation, to form single coacervate droplets within lipid vesicles. The process is completely reversible as coacervate droplets can be disassembled by increasing the pH above the pK_a. We further show that pH-triggered coacervation in the presence of low concentrations of enzymes activates dormant enzyme reactions by increasing the local concentration within the coacervate droplets and changing the local environment around the enzyme. In conclusion, this work establishes a tunable, pH responsive, enzymatically active multi-compartment synthetic cell. The system is readily transferred into microfluidics, making it a robust model for addressing general questions in biology, such as the role of phase separation and its effect on enzymatic reactions using a bottom-up synthetic biology approach.     

An Amperometric Glucose Biosensor Based on Poly (Pyrrole‐2‐Carboxylic Acid)/Glucose Oxidase Biocomposite

A newly developed amperometric glucose biosensor based on graphite rod (GR) working electrode modified with biocomposite consisting of poly (pyrrole‐2‐carboxylic acid) (PCPy) particles and enzyme glucose oxidase (GOx) was investigated. The PCPy particles were synthesized by chemical oxidative polymerization technique using H₂O₂ as initiator of polymerization reaction and modified covalently with the GOx (PCPy‐GOx) after activation of carboxyl groups located on the particles surface with a mixture of N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS). Then the PCPy‐GOx biocomposite was dispersed in a buffer solution containing a certain amount of bovine serum albumin (BSA). The resulting biocomposite suspension was adsorbed the on GR electrode surface with subsequent solvent airing and chemical cross‐linking of the proteins with glutaraldehyde vapour (GR/PCPy‐GOx). It was determined that the current response of the GR/PCPy‐GOx electrodes to glucose measured at +300 mV vs Cl reference electrode was influenced by the duration of the PCPy particles synthesis, pH of the GOx solution used for the PCPy particles modification and the amount of immobilized PCPy‐GOx biocomposite. An optimal pH of buffer solution for operation of the biosensor was found to be 8.0. Detection limit was determined as 0.039 mmol L⁻¹ according signal to noise ratio (S/N: 3). The proposed glucose biosensor was tested in human serum samples.     

Carbon Nanostructured Screen‐printed Electrodes Modified with CuO/Glucose Oxidase/Maltase/SiO2 Composite Film for Maltose Determination

A sensitive voltammetric method was developed to determine maltose in beverage products using a carbon nanostructured screen‐printed electrode modified with CuO/glucose oxidase/maltase/SiO₂ biocomposite film. Adding CuO particles was done to possess catalytic activity toward hydrogen peroxide. Electrode modified by glucose oxidase and maltase shows a good response to maltose. A well‐defined reduction peak was registered at the potential of ?0.55 V (vs. Ag/AgCl) which intensity increases linearly with the concentration of maltose ranging from 0.01 to 0.1 mmol L^?1. The calculated limit of detection was 0.005 mmol L^?1. Tested on the beer samples, the developed CuO/glucose oxidase/maltase/SiO₂ biocomposite film covered carbon nanostructured screen‐printed electrode is showed to be a prospective sensitive element of the third generation biosensor for maltose.     

pH‐Switchable Bioelectrocatalysis Based on Weak Polyelectrolyte Multilayers

Weak polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were assembled into {PAH/PAA}_n layer‐by‐layer films on electrodes. The cyclic voltammetry (CV) response of ferrocenecarboxylic acid (Fc(COOH)) at {PAH/PAA}₅ film electrodes assembled under the specific condition showed pH‐sensitive “on‐off” switching property. This property was further used to control the electrocatalytic oxidation of glucose by glucose oxidase (GOD) with Fc(COOH) as the electron transfer mediator, so that the pH‐switchable bioelectrocatalysis could be realized. The mechanism of pH‐sensitive behavior of the system was explored and believed to originate from the pH‐dependent structure change of the films.     

The Role of Chemically Innocent Polyanions in Active,Chemically Fueled Complex Coacervate Droplets

Complex coacervation describes the liquid-liquid phase separation of oppositely charged polymers. Active coacervates are droplets in which one of the electrolyte's affinity is regulated by chemical reactions. These droplets are particularly interesting because they are tightly regulated by reaction kinetics. For example, they serve as a model for membraneless organelles that are also often regulated by biochemical transformations such as post-translational modifications. They are also a great protocell model or could be used to synthesize life–they spontaneously emerge in response to reagents, compete, and decay when all nutrients have been consumed. However, the role of the unreactive building blocks, e.g., the polymeric compounds, is poorly understood. Here, we show the important role of the chemically innocent, unreactive polyanion of our chemically fueled coacervation droplets. We show that the polyanion drastically influences the resulting droplets′ life cycle without influencing the chemical reaction cycle–either they are very dynamic or have a delayed dissolution. Additionally, we derive a mechanistic understanding of our observations and show how additives and rational polymer design help to create the desired coacervate emulsion life cycles.