Abstract
Cells rely upon producing enzymes at precise rates and stoichiometry for maximizing functionalities. The reasons for this optimal control are unknown, primarily because of the interconnectivity of the enzymatic cascade effects within multi‐step pathways. Here, an elegant strategy for studying such behavior, by controlling segregation/combination of enzymes/metabolites in synthetic cell‐sized compartments, while preserving vital cellular elements is presented. Therefore, compartments shaped into polymer GUVs are developed, producing via high‐precision double‐emulsion microfluidics that enable: i) tight control over the absolute and relative enzymatic contents inside the GUVs, reaching nearly 100% encapsulation and co‐encapsulation efficiencies, and ii) functional reconstitution of biopores and membrane proteins in the GUVs polymeric membrane, thus supporting in situ reactions. GUVs equipped with biopores/membrane proteins and loaded with one or more enzymes are arranged in a variety of combinations that allow the study of a three‐step cascade in multiple topologies. Due to the spatiotemporal control provided, optimum conditions for decreasing the accumulation of inhibitors are unveiled, and benefited from reactive intermediates to maximize the overall cascade efficiency in compartments. The non‐system‐specific feature of the novel strategy makes this system an ideal candidate for the development of new synthetic routes as well as for screening natural and more complex pathways.
In living cells, there exists an incredible variety of sequential chains of reactions. These so‐called reaction pathways are catalyzed by mutually compatible and selective enzymes, which perform central functions, such as breaking down toxins,[1] converting nutrients into energy,[2] or duplicating DNA.[3] To optimize these pathways and thus to guarantee their maximum chance of survival, cells produce enzymes at precise and constant rates.[4] However, because of cellular complexity, determination of optimal enzyme levels is still unclear, in particular because specific enzymatic functions are strongly interconnected with their cascade effects.[5] A smart manner to study the behavior of enzymes, when involved in complex reactions, consists of taking advantage of synthetic micrometer‐sized compartments shaped into spherical architectural bodies.[6–8] Though these idealized models favor even partitioning of membrane proteins and simplify diffusion gradients, similarly to cells, they provide the desired membrane selectivity and permeability.[9, 10]
Single specific functions have been presented by combining enzymes (the catalytic compounds) with synthetic supramolecular assemblies, either intrinsically semipermeable, such as lipid‐coated porous silica particles,[11] protein cages,[12] layer‐by‐layer capsules,[13–15] and polydopamine capsules,[16, 17] or rendered permeable by the insertion of biopores/membrane proteins (acting as ”gates” for the passage of molecules), as in lipid‐based[7, 18, 19] or polymer‐based[8, 20–22] giant unilamellar vesicles (GUVs).
At present, GUVs formed with amphiphilic block copolymers are particularly appealing because they provide enhanced structural membrane properties compared to lipids, since they possess compartments with higher chemical versatility, controlled permeability, robustness, and stability.[23] Nevertheless, common approaches to form polymer GUVs by self‐assembly, as electroformation[24] and film‐rehydration,[25] rely on the statistical process of encapsulation of biomolecules with a probability of finding the designed amount of one type of enzyme inside the compartments ranging from 12–57%. In the context of elementary biochemical pathways where at least two enzymes are present, this scenario is even more unsatisfactory, with co‐encapsulation of two enzymes inside one compartment as low as 10–22%.[26, 27] These shortcomings are worsened by the fact that these probabilities have a large uncertainty induced by specific properties of enzymes (e.g., solubility and stability). To date, scientists have struggled to work with state‐of‐the‐art average values for number/mass of enzymes that are vastly non‐representative (limited by a small sample size) and extremely low, due to severe dilution as a result of the self‐assembly processes.[28, 29]
Double emulsion microfluidics has a significant role to play in the fine control of these parameters (encapsulated content and membrane compositions), coupled with the capability for high‐throughput and on‐demand generation.[30] However, while double emulsions have been extensively used to form GUVs for programmed release of encapsulated hydrophilic[31–33] and/or hydrophobic[31, 34] cargos, their capabilities are still undeveloped for the study of enzymatic pathways, where they are expected to have substantial advantages. In this respect, double emulsions can serve as templates for producing ideal cell‐sized compartments with precise control of properties, such as: i) size (as per design) and extremely narrow size distribution; ii) internal biomolecular content and distribution; iii) membrane organization, that is, synthetic membrane composition and insertion of peptides/membrane proteins; and ultimately, iv) features for enzymatic reactions and pathway signaling.
Here, we introduce a novel strategy for studying multi‐step enzymatic reactions inside tailored synthetic compartments, systematically arranged in a variety of combinatorial configurations. These compartments in the form of polymer GUVs have been produced via the high precision double emulsion technique, which allows the vital control of the amount/number/ratio of encapsulated enzymes. Moreover the technique has been optimized to render the membrane of the GUVs permeable for diffusion of enzymatic substrates/products in and out of the compartments to support in situ enzymatic reactions. To obtain GUVs with essential properties for insertion of peptides/membrane proteins that make their membranes permeable, we selected amphiphilic block copolymers, based on poly(dimethylsiloxane) (PDMS) and poly(2‐methyl‐2‐oxazoline) (PMOXA) as the corresponding hydrophobic and hydrophilic domains, which have already been used to produce GUVs with the required fluidity and flexibility.[35] Together with the enzymes encapsulated inside the GUVs, equipping GUVs with biopores/membrane proteins allows investigation of multi‐step cascade reactions via the spatial segregation/combination of various enzyme types in different compartments. A three‐step cascade reaction, based on well‐known enzymes, was selected as a model to provide evidence on how the overall reaction can be affected by compartmentalization and single enzyme behavior. A combinatorial strategy based on a plethora of possible segregation configurations, for example, with enzymes separated from one another or in several combinations of encapsulation and co‐encapsulation scenarios, was facilitated by the straightforward manner in which double emulsion microfluidics controls the production of the enzyme‐loaded GUVs. Thanks to the constant enzyme molar ratios provided by our method, effects, such as the proximity of enzyme and metabolites, and molecular diffusion through membranes have been decoupled, thus allowing limiting factors to be understood and cascades optimized. This fine spatial control provides optimum conditions for reducing competing side reactions, decreasing the accumulation of inhibitory or reactive intermediates, and ultimately increasing reaction rates by maximizing the cascade productivity.
Furthermore, this strategy allows the steps for studying and optimizing complex cascade reactions to be established in a controlled space and time manner, without involving the difficult and time consuming labor of varying enzymatic concentrations. Thus, our combinatorial study represents a system‐independent tool for coordinating multi‐step pathways, with complexity closer to that of cells for exploring new domains of application.
In the literature, two separate approaches have been used for the creation of double emulsion templated GUVs in microfluidic devices, using glass capillaries or molded in PDMS. Each one has its own advantages and limitations. Glass capillaries have been widely used for production of different types of capsules,[32, 36, 37] including GUVs with ultra thin shells,[38, 39] but the device production and operation are quite challenging: If capillaries are misaligned or not well sanded, instabilities can occur and double emulsions might not be produced because of uncontrolled encounter of the fluids.[40] Reproducible fabrication of microfluidic devices is more easily achieved if they are made from masks and molds, which is predominantly the case when soft lithography is employed to fabricate microchips made from PDMS.[41] However, despite rapid prototyping and easy replication, there are difficulties in modifying the chemical properties of PDMS surfaces to tune their wettability toward different fluids.[38] Forming double emulsions in these conditions requires the oil phase enveloping the inner droplets to be thicker to avoid their collapse, and this may prevent assembly of the GUVs.[42] Thus, to obtain GUVs with a homogeneous polymeric membrane that favors biopore/membrane protein insertion, a higher level of precision and reproducibility is required for generating double emulsions with thin organic shells, which neither glass capillaries nor PDMS microdevices alone can in the long run provide. For this reason, we now combine the best characteristics of both aforementioned approaches to produce novel microfluidic devices that are based on high‐resolution solid‐state manufacturing and aim at exploiting the high chemical and mechanical compatibility of silicon‐glass devices. For that purpose, channels were etched into a silicon (Si) substrate using deep‐reactive ion etching (DRIE) (that provides rectangular cross‐sections with a desired depth, described in Supporting Information), and were closed with glass covers by anodically bonding them to the Si substrate. Both the native oxide of the Si bottom channel walls as well as the glass channel top wall offer universal solvent compatibility (avoiding channel swelling) and multiple ways to control surface chemistry. Furthermore, the architecture provides a high mechanical robustness, in particular compared to PDMS‐based devices, which prevents oscillatory ”breathing” of the channels upon operation, preserving the geometry of the junction, important for double‐emulsion generation.[43]…..
