Aqueous Droplets Research Articles

Generation of polyethylene glycol-dextran aqueous two-phase system droplets using different culture media under in vitro conditions

Aqueous two-phase systems (ATPS) involve the combination of two immiscible polymers that at certain concentrations form a biphasic separation system. Due to the physicochemical properties and the low interfacial tension of the ATPS-forming polymers, the generation of ATPS droplets began to be explored. This work aims to characterize the generation of ATPS droplets evaluating four polyethylene glycol (PEG)-dextran (DEX) systems (combinations of molecular weights and concentrations) using Essential 8 (Gibco), DMEM-F12 (Gibco), StemLine II (Sigma-Aldrich), MSCs Basal (ATCC), RPMI 1640 (Gibco), and NutriStem (Sartorius) as ATPS solvents under in vitro conditions. In 96 well-plates, one DEX droplet of 1 μL was immersed at the bottom of the well filled with 50 μL of PEG bulk phase. ATPS droplets were generated in all tested PEG-DEX systems showing circular morphology with a mid-range diameter size in the six-culture media of 1.30 ± 0.18 mm, 1.30 ± 0.22 mm, 1.30 ± 0.43 mm, 1.21 ± 0.27 mm, 1.20 ± 0.16 mm, and 1.07 ± 0.21 mm, respectively. Uniformity was susceptible to the culture media composition showing a coefficient of variation range of 0.64–12.40 % among the four studied PEG-DEX systems. By controlling the geometry of ATPS droplets, specific cell patterns can be determined for the further construction of different cell microenvironments in 3D cultures using ATPS.

Multifunctional Droplets Formed by Interfacially Self-Assembled Fluorinated Magnetic Nanoparticles for Biocompatible Single Cell Culture and Magnet-Driven Manipulation.

The ability to encapsulate and manipulate droplets with a picoliter volume of samples and reagents shows great potential for practical applications in chemistry, biology, and materials science. Magnetic control is a promising approach for droplet manipulation due to its ability for wireless control and its ease of implementation. However, it is challenged by the poor biocompatibility of magnetic materials in aqueous droplets. Moreover, current droplet technology is problematic because of the molecule leakage between droplets. In the paper, we propose multifunctional droplets with the surface coated by a layer of fluorinated magnetic nanoparticles for magnetically actuated droplet manipulation. Multifunctional droplets show excellent biocompatibility for cell culture, nonleakage of molecules, and high response to a magnetic field. We developed a strategy of coating the F-MNP@SiO2 on the outer surface of droplets instead of adding magnetic material into droplets to enable droplets with a highly magnetic response. The encapsulated bacteria and cells in droplets did not need to directly contact with the magnetic materials at the outer surface, showing high biocompatibility with living cells. These droplets can be precisely manipulated based on magnet distance, the time duration of the magnetic field, the droplet size, and the MNP composition, which well match with theoretical analysis. The precise magnetically actuated droplet manipulation shows great potential for accurate and sensitive droplet-based bioassays like single cell analysis.

Signal amplification strategies for optical biodetection at the liquid crystal–solid interface

ABSTRACTLiquid crystals (LCs) are reliable signal transducers for biosensors with chemical and biological sensing potentials demonstrated on the basis of their anisotropy. Thanks to the high sensitivity and fast response to analytes, the LC–solid, LC–aqueous and LC droplet interfaces facilitate sensing platforms to be established without the requirement of sophisticated and costly analytical instruments. In this mini-review, we summarise strategies of optical signal amplification to improve the detection limit of biological analytes at the LC–solid interface. Because approaches towards signal amplification for a single target analyte are seldom compared by discussing various LC-based biosensing platforms, this mini-review may provide implications for integrating current technologies to promote the development of multiplex platforms based on the LC–solid interface.

Uncovering Bacterial Hosts of Class 1 Integrons in an Urban Coastal Aquatic Environment with a Single-Cell Fusion-Polymerase Chain Reaction Technology.

Horizontal gene transfer (HGT) is a key driver of bacterial evolution via transmission of genetic materials across taxa. Class 1 integrons are genetic elements that correlate strongly with anthropogenic pollution and contribute to the spread of antimicrobial resistance (AMR) genes via HGT. Despite their significance to human health, there is a shortage of robust, culture-free surveillance technologies for identifying uncultivated environmental taxa that harbor class 1 integrons. We developed a modified version of epicPCR (emulsion, paired isolation, and concatenation polymerase chain reaction (PCR)) that links class 1 integrons amplified from single bacterial cells to taxonomic markers from the same cells in emulsified aqueous droplets. Using this single-cell genomic approach and Nanopore sequencing, we successfully assigned class 1 integron gene cassette arrays containing mostly AMR genes to their hosts in coastal water samples that were affected by pollution. Our work presents the first application of epicPCR for targeting variable, multigene loci of interest. We also identified the Rhizobacter genus as novel hosts of class 1 integrons. These findings establish epicPCR as a powerful tool for linking taxa to class 1 integrons in environmental bacterial communities and offer the potential to direct mitigation efforts toward hotspots of class 1 integron-mediated dissemination of AMR.

A novel droplet-based approach to study phase transformations in lyotropic liquid crystalline systems

HypothesisLyotropic liquid crystals (LLC) and their phase transformations in response to stimuli have gathered much interest for controlled and ‘on-demand’ drug applications. Bulk methods of preparation impose limitations on studying the transformations, especially induced by compositional changes, such as enzymatic changes to lipid structure. Here we hypothesise that controlled microfluidic production and coalescence of dissimilar aqueous and lipid droplets emulsified in a third mutually immiscible liquid will provide a new approach to the spatio-temporal study of structure formation in lyotropic liquid crystalline materials. ExperimentsSeparate lipid and aqueous droplets, dispersed in a fluorocarbon oil were generated using a microfluidic format. The chip, prepared as a hybrid polydimethylsiloxane (PDMS) and glass microfluidic device, was constructed to enable in-situ acquisition of time-resolved synchrotron small angle X-ray scattering (SAXS) and crossed polarised light microscopy of the coalesced droplets to determine the structures present during aging. FindingsJanus-like droplets formed upon coalesce, with distinct lipid and aqueous portions with a gradient between the two sides of the merged droplet. SAXS and polarised light microscopy revealed a progression of mesophases as the lipid portion was hydrated by the aqueous portion via the diffusion limited interface which separated the portions. Thus demonstrating, on a droplet scale, a new approach for studying the phase transformation kinetics and identification of non-equilibrium phase in droplet-based lyotropic liquid systems.

Evaluation of Analyte Transfer between Microfluidic Droplets by Mass Spectrometry.

Droplet microfluidics enables high-throughput experimentation and screening by encapsulating chemical and biochemical samples in aqueous droplets segmented by an immiscible fluid. In such experiments, it is critical that each droplet remains chemically distinct. A common approach is to use fluorinated oils with surfactants to stabilize droplets. However, some small molecules have been observed to transport between droplets under these conditions. Attempts to study and mitigate this effect have relied on evaluating crosstalk using fluorescent molecules, which inherently limits the analyte scope and conclusions drawn about the mechanism of the effect. In this work, transport of low molecular weight compounds between droplets was investigated using electrospray ionization mass spectrometry (ESI-MS) for measurement. The use of ESI-MS significantly expands the scope of analytes that can be tested. We tested 36 structurally diverse analytes that were found to exhibit crosstalk ranging from negligible to complete transfer using HFE 7500 as the carrier fluid and 008-fluorosurfactant as a surfactant. Using this data set, we developed a predictive tool showing that high log P and log D values correlate with high crosstalk, and high polar surface area and log S correlate with low crosstalk. We then investigated several carrier fluids, surfactants, and flow conditions. It was discovered that transport is strongly dependent on all of these factors and that experimental design and surfactant tailoring can reduce carryover. We present evidence for mixed crosstalk mechanisms including both micellar and oil partitioning transfer. By understanding the driving mechanisms, surfactant and oil compositions can be designed to better reduce chemical transport for screening workflows.

Facile synthesis of chromium chloride/poly(methyl methacrylate) core/shell nanocapsules by inverse miniemulsion evaporation method and application as delayed crosslinker in secondary oil recovery

Cr(III)–hydrolyzed polyacrylamide (HPAM) gels have been extensively studied as a promising strategy controlling waste water production for mature oilfields. However, the gelation time of the current technologies is not long enough for in-depth placement. In this study, we report a novel synthesis method to obtain chromium chloride/poly(methyl methacrylate) (PMMA) nanocapsules which can significantly delay the gelation of HPAM through encapsulating the chromium chloride crosslinker. The chromium chloride-loaded nanocapsules (Cr–NC) are prepared via a facile inverse miniemulsion evaporation method during which the hydrophobic PMMA polymers, pre-dispersed in an organic solvent, were carefully controlled to precipitate onto stable aqueous miniemulsion droplets. The stable aqueous nanodroplets (W) containing Cr(III) are dispersed in a mixture of organic solvent (O 1 ) with PMMA and nonsolvent medium (O 2 ) to prepare an inverse miniemulsion. With the evaporation of the O 1 , PMMA forms Cr–NCs around the aqueous droplets. The Cr–NCs are readily transferred into water from the organic nonsolvent phase. The Cr–NCs exhibit the tunable size (358–983 nm), Cr loading (7.1–19.1%), and Cr entrapment efficiency (11.7%–80.2%), with tunable zeta potentials in different PVA solution. The Cr–NCs can delay release of Cr(III) and prolong the gelation time of HPAM up to 27 days.

High-speed imaging of giant unilamellar vesicle production in cDICE.

Giant unilamellar vesicles (GUVs) are cell-sized containers that are commonly used as three-dimensional model membranes in biophysics, as in vitro model systems in synthetic biology, and even as cargo carriers in various other research fields. Despite their ubiquitous use, there is still no one-size-fits-all GUV production method. Over the years, numerous methods have been developed, attempting to meet the demanding requirements of robustness, reliability, and high yield while simultaneously achieving robust encapsulation. Double emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE) that make use of this principle, have gained popularity in recent years. In cDICE, aqueous droplets that originate from a capillary orifice are continuously forced through an oil-water interface by centrifugal force, thereby forming a lipid bilayer and thus GUVs. Although cDICE and related methods are frequently used in the field, the complexity of the underlying principles and fluid dynamics has not been considered previously, and how exactly the GUVs are being formed remains unknown. To elucidate the process of GUV formation in cDICE, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real time. We focused on the capillary orifice, where initial droplet formation occurs, and on the oil-water interface, where droplets are converted into GUVs. Our experiments reveal a complex droplet formation process at the capillary orifice and suboptimal droplet transfer through the water-oil interface, which we explain using fluid dynamics and theoretical modeling. Our results are a first step towards explaining the widely observed variation in encapsulation efficiency and size polydispersity in cDICE. Ultimately, these results will contribute to a better understanding of GUV formation processes in cDICE and in extension, in double emulsion-based methods in general.

Electrotaxis behavior of droplets composed of aqueous Belousov-Zhabotinsky solutions suspended in oil phase

Taxis is ubiquitous in biological and physical chemistry systems as a response to various external stimulations. We prepared aqueous droplets containing Belousov-Zhabotinsky (BZ) solutions suspended on an oleic acid oil phase subject to DC electric field and found that these BZ droplets undergo chemically driven translational motion towards the negative electrode under DC electric field. This electrotaxis phenomenon originates from the field-induced inhomogeneous distribution of reactants, in particular Br^{-} ions, and consequently the biased location of the leading centers towards the positive electrode. We define the ’leading center’ (LC) as a specific location within the droplet where the BZ chemical wave (target pattern) is initiated. The chemical wave generated from the LC propagates passing the droplet center of mass and creates a gradient of interfacial tension when reaching the droplet-oil interface on the other side, resulting in a momentum exchange between the droplet and oil phases which drives the droplet motion in the direction of the electric field. A greater electric field strength renders a more substantial electrotaxis effect.

Preparation of Hydrophobic Monolithic Supermacroporous Cryogel Particles for the Separation of Stabilized Oil-in-Water Emulsion

Here, we prepared hydrophobic cryogel particles with monolithic supermacropores based on poly-trimethylolpropane trimethacrylate (pTrim) by combining the inverse Leidenfrost effect and cryo-polymerization technique. The hydrophobic cryogel particles prepared by adopting this method demonstrated the separation of the stabilized O/W emulsion with surfactant. The prepared cryogel particles were characterized in terms of macroscopic shape and porous structure. It was found that the cryogel particles had a narrow size distribution and a monolithic supermacroporous structure. The hydrophobicity of the cryogel particles was confirmed by placing aqueous and organic droplets on the particles. Where the organic droplet was immediately adsorbed into the particles, the aqueous droplet remained on the surface of the particle due to repelling force. In addition, after it adsorbed the organic droplet the particle was observed, and the organic solvent was diffused into the entire particle. It was indicated that monolithic pores were distributed from the surface to the interior. Regarding the application of the hydrophobic cryogel particles, we demonstrated the separation of a stabilized oil-in-water emulsion, resulting in the successful removal of the organic solvent from the emulsion.

Four-petal aqueous imbibition into woven cloth

HypothesisImproving the processing efficiency of aerosol-coating technologies during mass production requires optimal nozzle spacing to allow complete surface coverage while at the same time not over-using the coating fluid. The difficult challenge is to estimate quantitatively the substrate coverage of fine droplets. Bouncing, splashing, and imbibition of droplets on solid surfaces have been widely explored, but little attention has been paid to liquid imbibition into woven textiles. ExperimentsHere, we experimentally and theoretically study the imbibition dynamics of aqueous droplets on woven cloths. The experimental process was observed using magnified visual observation. A proposed continuum mathematical model well predicts the aqueous imbibition fronts as a function of time. FindingsA captivating four-petal imbibition spreading pattern is observed at enhanced magnification. The imbibition occurs separately in the megapores of the cloth between yarns, and in smaller minipores within individual yarn bundles. Surprisingly, weave intersections do not allow cross imbibition accentuating an anisotropic imbibition pattern. The proposed model achieves quantitative agreement with experiment. This is the first time that the mechanisms of four-petal droplet deposition, spreading, and imbibition into woven cloth have been outlined and successfully simulated. The mathematical model predicts advancement of liquids in anisotropic woven cloth, and permits evaluation of the coverages of droplet spreading.

A Molecular Dynamic Study of the Effects of Surface Partitioning on the OH Radical Interactions with Solutes in Multicomponent Aqueous Aerosols

The surface-bulk partitioning of small saccharide and amide molecules in aqueous droplets was investigated using molecular dynamics. The air-particle interface was modeled using a 80 Å cubic water box containing a series of organic molecules and surrounded by gaseous OH radicals. The properties of the organic solutes within the interface and the water bulk were examined at a molecular level using density profiles and radial pair distribution functions. Molecules containing only polar functional groups such as urea and glucose are found predominantly in the water bulk, forming an exclusion layer near the water surface. Substitution of a single polar group by an alkyl group in sugars and amides leads to the migration of the molecule toward the interface. Within the first 2 nm from the water surface, surface-active solutes lose their rotational freedom and adopt a preferred orientation with the alkyl group pointing toward the surface. The different packing within the interface leads to different solvation shell structures and enhanced interaction between the organic molecules and absorbed OH radicals. The simulations provide quantitative information about the dimension, composition, and organization of the air-water interface as well as about the nonreactive interaction of the OH radicals with the organic solutes. It suggests that increased concentrations, preferred orientations, and decreased solvation near the air-water surface may lead to differences in reactivities between surface-active and surface-inactive molecules. The results are important to explain how heterogeneous oxidation mechanisms and kinetics within interfaces may differ from those of the bulk.

Terahertz refractive phenotype of living cells.

Cellular refractive index is a vital phenotypic parameter for understanding the cell functional activities. So far, there remains technical challenges to obtain refractive index of viable cells at the terahertz frequency in which contains rich information closely related to their physiological status. Here we introduce a label-free optical platform for interrogating cellular phenotypes to measure the refractive index of living cells in near-physiological environments by using terahertz spectroscopy with the combination of cellular encapsulation in a confined solution droplet. The key technical feature with cells encapsulated in aqueous droplets allows for keeping cellular viability while eliminating the strong adsorption of solvent water to terahertz signal. The obtained high signal-to-noise ratio enables to differentiate different cell types (e.g., E. coli, stem cell and cancer cell) and their states under stress conditions. The integrating of terahertz spectroscopy to droplet microfluidic further realizes automated and high-through sample preparation and detection, providing a practical toolkit for potential application in cellular health evaluation and phenotypic drug discovery.

Controlled release of microcargo from water-in-liquid crystal emulsions via interfacial shear induced by synthetic microstirrers.

Past studies demonstrated that the microcargo carrying aqueous droplets trapped in LCs through elastic stresses can be triggered to release by applying shear to LC-bulk interfaces. Herein, we report our investigations on the release mechanisms of such microcargo entrapped in aqueous droplets of W-in-LC emulsions via interfacial shear caused by the synthetic micro-stirrer microparticle assemblies of iron oxide particles rotated with a magnetic field. We show that a three fold control over the release rate of the tracer molecules is possible that have initial release rates of 14.3 ± 2.5 μg cm-2 h-1, 26.5 ± 3.4 μg cm-2 h-1, and 46.9 ± 4.6 μg cm-2 h-1 when the rotation of magnetic flux was 250, 500 and 1000 rpm, respectively. We measure the release rates to reduce to 5.4 ± 1.2 μg cm-2 h-1, 6.3 ± 0.7 μg cm-2 h-1, and 20.0 ± 3.2 μg cm-2 h-1 after 60 min of shearing at 250, 500 and 1000 rpm, respectively. We present evidence for the release mechanism resulting in such temporal release profiles that correlate with the magnitude of the shear applied to the interface, interfacial coverage of the microstirrers, and the creaming effect of the aqueous droplets doped with tracers. We also report that the influence of the interfacial density of the microparticles that showed an intermediate areal density is required to achieve a maximized release rate. Additionally, we found evidence of the influence of the droplet charge that critically determines the release. This study highlights the importance of the colloidal and interfacial phenomena in the release profiles of the dispersed droplets present in a LC medium.

One step generation of single-core double emulsions from polymer-osmose-induced aqueous phase separation in polar oil droplets.

Water-in-oil-in-water emulsions (W/O/W) are aqueous droplet(s) embedded within oil droplets dispersed in a continuous water phase. They are attracting interest due to their possible applications from cosmetic to food science since both hydrosoluble and liposoluble cargos can be encapsulated within. They are generally prepared using a one-step or a two-step method, phase inversion and also via spontaneous emulsification. Here, we describe a general and simple one-step method based on hydrophilic polymers dispersed in polar oils to generate osmose-induced diffusion of water into oil droplets, forming polymer-rich aqueous droplets inside the oil droplets. Polyethylene glycol, but also other hydrophilic polymers (branched polyethylene imine or polyvinyl pyrrolidone) were successfully dispersed in 1-octanol or other polar oils (oleic acid or tributyrin) to produce an O/W emulsion that spontaneously transformed into a W1/O/W2 emulsion, with the inner aqueous droplet (W1) only containing the hydrophilic polymer initially dispersed in oil. By combining single drop experiments, with macroscopic viscosity measurements, we demonstrated that the double emulsion resulted of water diffusion, which amplitude could be adjusted by the polymer concentration. The production of high internal phase emulsions was also achieved, together with a pH-induced transition from multiple to single core double emulsion. We expect this new method for producing double emulsions to find applications in domains of microencapsulation and materials chemistry.

Smart Super Hydrophobic Textiles Utilizing Long-Range Antenna Sensor for Hazardous Aqueous Droplet Detection and Prevention

This review paper explores the integration of smart superhydrophobic textiles and antenna sensors for detecting and preventing hazardous aqueous droplets. Superhydrophobic surfaces have gained attention for their water-repellent properties, and advancements in fabrication techniques have enabled the creation of superhydrophobic textiles that effectively repel liquid droplets. To enhance functionality, antenna sensors are incorporated into the fabric structure, allowing real-time detection of toxic chemicals, pathogens, and other contaminants present in aqueous droplets. The integration of antenna sensors with superhydrophobic textiles presents a synergistic approach to addressing challenges in detection and prevention in healthcare, industrial safety, and environmental monitoring. The review covers recent advancements in materials, fabrication techniques, and water-repellent mechanisms of superhydrophobic textiles, as well as the integration strategies for sensor implementation. It also discusses potential applications, limitations, and future prospects. The findings emphasize the promising opportunities for these technologies in improving safety measures, monitoring capabilities, and rapid response mechanisms across various industries. Overall, the integration of smart superhydrophobic textiles and antenna sensors holds great potential for revolutionizing the detection and prevention of hazardous aqueous droplets.

Measurement and Prediction of Droplet Entrainment in Inclined Liquid–Liquid Flow by PLIF Method

Inclined liquid-liquid two-phase flows widely exist in petroleum, chemical, and other important industrial processes. The study of droplet entrainment in inclined flow is of great significance for investigating the heat/mass transfer between phases and optimizing the industrial production processes. In this study, a planar laser-induced fluorescence (PLIF) system is designed to visualize the inclined liquid-liquid flows. The silicone oil (organic phase) and water/glycerol mixture (aqueous phase) are used as the experimental media. The length and amplitude of the interfacial waves are derived from the flow visualizations. The relationship between the critical wavelengths and Froude number is explored to indicate the transition from stratified flow (ST) to stratified flow with mixing at the interface (ST&MI). The wave aspect ratio is derived to characterize the instability and breakage of the interfacial wave. Meanwhile, the entrainment ratios of dispersed droplets in the continuous phases are detected, and the effects of the flow rate and the interfacial shear on the droplet entrainment are studied. Finally, a physical model is developed to predict the droplet entrainment ratios based on the force balance analysis of the interfacial wave. In general, the developed model is effective in predicting the droplet entrainment ratio with mean absolute errors of 0.015 and 0.023 for the organic and aqueous droplets, respectively.

Ion-modulated interfacial fluorescence in droplet microfluidics using an ionophore-doped oil.

Fluorescence at the oil-water interface is used for chemical sensing in droplet microfluidics. Potassium ions in aqueous droplets are extracted into oil segments doped with an ionophore, a cation exchanger, and a cationic dye to expel the dye. When a low concentration of dye with a balanced solubility is used, it actively accumulates at the thin interface between oil and water instead of getting dissolved in the aqueous phase. The interfacial fluorescence is monitored distinct from the fluorescence in the oil sensor and the aqueous sample, allowing for highly sensitive and selective turn-on fluorescence sensing of ions.

Does liquid-liquid phase separation impact ice nucleation in mixed polyethylene glycol and ammonium sulfate droplets?

Particles can undergo different phase transitions in the atmosphere including deliquescence, liquid-liquid phase separation (LLPS), melting, and freezing. In this study, phase transitions of particles/droplets containing polyethylene glycol with a molar mass of 400 g mol-1 (PEG400) and ammonium sulfate (AS), i.e., PEG400-AS particles/droplets, were investigated at different organic-to-inorganic dry mass ratios (OIRs) under typical tropospheric temperatures and water activities (aw). The investigated droplets (60-100 μm) with or without LLPS in the closed system froze through homogeneous ice nucleation. At temperatures lower than 200 K, multiple ice nucleation events were observed within the same individual droplets at low aw. Droplets with and without LLPS shared similar lambda values at the same OIR according to the lambda approach indicating they form ice through the same mechanism. A parameterization of lambda values was provided which can be used to predict freezing temperature of aqueous PEG400-AS droplets. We found that adding AS reduces the temperature dependence of aw in aqueous PEG400 droplets. Assuming incorrectly that aw is temperature-independent for a constant droplet composition leads to a deviation between the experimental determined ice nucleation rate coefficients for droplets at OIR > 1 and the predicted values by the water-activity-based ice nucleation theory. We proposed a parameterization of temperature dependence of aw to minimize the deviations of the measured melting temperatures and nucleation rate coefficients from the corresponding predictions for aqueous PEG400-AS system.

Multicompartment calcium alginate microreactors to reduce substrate inhibition in enzyme cascade reactions.

The formation of macromolecularly enriched condensates through associative or segregative liquid-liquid phase separation phenomena is known to play a central role in controlling various cellular functions in nature. The potential to spatially and temporally modulate multistep chemical reactions and pathways has inspired the use of phase-separated systems for the development of various synthetic colloidal micro- and nanoreactor systems. Here, we report a rational and synthetically minimal design strategy to emulate intended spatiotemporal functions in morphologically intricate and structurally defined calcium alginate hydrogel microreactors possessing multicompartmentalized internal architectures. Specifically, we implement a thermal phase separation protocol to achieve fine-control over liquid-liquid phase separation inside complex aqueous emulsion droplet templates that are loaded with hydrophilic polymer mixtures. Subsequent gelation of alginate-containing droplet templates using a novel freeze-thaw approach that can be applied to both scalable batch production or more precise microfluidic methods yields particle replicas, in which subcompartmentalized architectures can be retained. Larger active components can be enriched in the internal compartments due to their preferential solubility, and we show that selective sequestration of enzymes serves to create desired microenvironments to control and tune the reaction kinetics of a multistep enzyme cascade by reducing their mutual interference. This demonstration of mitigating substrate inhibition that is based primarily on optimizing the multicompartmentalized hydrogel particle morphology offers new opportunities for the simple and synthetically-minimal batch generation of hydrogel-based synthesis microreactors.