Active Pulsatile Gels: From a Chemical Microreactor to a Polymeric Actuator.

We report on a synthesis protocol, experimental characterization, and theoretical modeling of active pulsatile Belousov-Zhabotinsky (BZ) hydrogels. Our two-step synthesis technique allows independent optimization of the geometry, the chemical, and the mechanical properties of BZ gels. We identify the role of the surrounding medium chemistry and gel radius for the occurrence of BZ gel oscillations, quantified by the Damköhler number, which is the ratio of chemical reaction to diffusion rates. Tuning the BZ gel size to maximize its chemomechanical oscillation amplitude, we find that its oscillatory strain amplitude is limited by the time scale of gel swelling relative to the chemical oscillation period. Our experimental findings are in good agreement with a Vanag-Epstein model of BZ chemistry and a Tanaka Fillmore theory of gel swelling dynamics.

Capsule-based colorimetric temperature monitoring system for customizable cold chain management.

The COVID-19 pandemic and the resulting supply chain disruption have rekindled crucial needs for safe storage and transportation of essential items. Despite recent advances, existing temperature monitoring technologies for cold chain management fall short in reliability, cost, and flexibility toward customized cold chain management for various products with different required temperature. In this work, we report a novel capsule-based colorimetric temperature monitoring system with precise and readily tunable temperature ranges. Triple emulsion drop-based microfluidic technique enables rapid production of monodisperse microcapsules with an interstitial phase-change oil (PCO) layer with precise control over its dimension and composition. Liquid-solid phase transition of the PCO layer below its freezing point triggers the release of the encapsulated payload yielding drastic change in color, allowing user-friendly visual monitoring in a highly sensitive manner. Simple tuning of the PCO layer's compositions can further broaden the temperature range in a precisely controlled manner. The proposed simple scheme can readily be formulated to detect both temperature rise in the frozen environment and freeze detection as well as multiple temperature monitoring. Combined, these results support a significant step forward for the development of customizable colorimetric monitoring of a broad range of temperatures with precision.

Robust and Reliable Fabrication of Gelatin Films Containing Micropatterned Opal Structures via Evaporative Deposition and Thermal Gelation.

Biopolymeric hydrogel materials containing tunable optical properties such as micropatterned artificial opal structures hold significant potential in various applications. Despite recent advances in fabrication techniques, simple, reliable, and tunable production of stimuli-responsive micropatterned opal hydrogels under mild conditions remains challenging. We report a simple micromolding-based evaporative deposition-thermal gelation technique for gelatin films that capture uniform opal micropatterns, aided by a potent aminopolysaccharide chitosan (CS) that provides binding affinity and structural stability. Our results show reliable, tunable, and high-fidelity fabrication of gelatin hydrogel films containing CS-opal micropatterns, while the as-prepared films show responsiveness to pH, ionic strength, and water content indicating a robust nature. Uniform CS-opal microparticles can also be readily prepared via removal of the gelatin through various simple routes, illustrating the crucial roles of CS and gelatin. We envision that this robust, reliable, and simple evaporative deposition-thermal gelation technique can be readily extended to prepare responsive biopolymeric materials for various applications.

Fine-tuning of MXene-nickel oxide-reduced graphene oxide nanocomposite bioelectrode: Sensor for the detection of influenza virus and viral protein.

Influenza viruses can cause epidemics through inter-human transmission, and the social consequences of viral transmission are incalculable. Current diagnostics for virus detection commonly relies on antibodies or nucleic acid as recognition reagent. However, a more advanced and general method for the facile development of new biosensors is increasing in demand. In this study, we report the fabrication of an ultra-sensitive peptide-based nanobiosensor using a nickel oxide (NiO)-reduced graphene oxide (rGO)/MXene nanocomposite to detect active influenza viruses (H1N1 and H5N2) and viral proteins. The sensing mechanism is based on the signal inhibition, the specific interaction between H1N1 (QMGFMTSPKHSV) and H5N1 (GHPHYNNPSLQL) binding peptides anchored on the NiO-rGO/MXene/glassy carbon electrode (GCE) surface and the viral surface protein hemagglutinin (HA) is the critical factor for the decrease in the peak current of the sensor. In this strategy, the NiO-rGO/MXene nanocomposite results in synergistic signal effects, including electrical conductivity, porosity, electroactive surface area, and active site availability when viruses are deposited on the electrode. Based on these observations, the results showed that the developed nanobiosensor was capable of highly sensitive and specific detection of their corresponding influenza viruses and viral proteins with a very low detection limit (3.63 nM of H1N1 and 2.39 nM for H5N1, respectively) and good recovery. The findings demonstrate that the proposed NiO-rGO/MXene-based peptide biosensor can provide insights for developing a wide range of clinical screening tools for detecting affected patients.

Triple Emulsion-Based Rapid Microfluidic Production of Core-Shell Hydrogel Microspheres for Programmable Biomolecular Conjugation.

We report a simple and rapid microfluidic approach to produce core-shell hydrogel microspheres in a single step. We exploit triple emulsion drops with sacrificial oil layers that separate two prepolymer phases, forming poly(ethylene glycol)-based core-shell microspheres via photopolymerization followed by spontaneous removal of the oil layer. Our technique enables the production of monodisperse core-shell microspheres with varying dimensions of each compartment by independently and precisely controlled flow rates. This leads to stable and uniform incorporation of functional moieties in the core compartment with negligible cross-contamination into the shell layer. Selective conjugation of biomolecules is enabled through a rapid bioorthogonal reaction with functional groups in the core compartment with minimal non-specific adsorption. Finally, in-depth protein conjugation kinetics studies using microspheres with varying shell porosities highlight the capability to provide tunable size-selective diffusion barriers by simple tuning of prepolymer compositions for the shell layer. Combined, these results illustrate a significant step forward for programmable high-throughput fabrication of multifunctional hydrogel microspheres, which possess substantial potential in a large array of biomedical and biochemical applications.

A Robust Fabrication Technique for Hydrogel Films Containing Micropatterned Opal Structures via Micromolding and an Integrated Evaporative Deposition-Photopolymerization Approach.

Opal-structured thin-film hydrogel materials with micropatterns hold great potential for utility in a wide range of sensing applications. Micropatterning offers key advantages such as ready addressability, high throughput assay, and multiplexing. However, controlled fabrication of such films in a rapid, inexpensive, and reliable manner remains a challenge. Existing techniques suffer from long opal deposition times and often involve complex and arduous steps. In this report, we examined a simple micromolding-based evaporation-polymerization method for the fabrication of poly(ethylene glycol)-based hydrogel films containing micropatterned opal structures. Specifically, intense and uniform opalescent colors were achieved by evaporative deposition of polystyrene bead solution in patterned micromolds. These opal micropatterns were then captured in hydrogel films by simple photopolymerization of a UV-curable PEG diacrylate solution. The as-prepared films show high tunability as well as responsiveness to various environmental cues readily manifested via shifts in color. Combined with UV-vis reflectance spectroscopy and scanning electron microscopy results, these findings illustrate the robust, simple, and reliable nature of our integrated deposition-polymerization approach for controlled fabrication of optically active and stimuli-responsive functional materials. We thus envision that the results and the facile approach reported here can be extended to many application areas including environmental monitoring, diagnostics, and biosensing applications.

Integrated Methods to Manufacture Hydrogel Microparticles Containing Viral-Metal Nanocomplexes with High Catalytic Activity.

Controlled synthesis of small and catalytically active noble metal nanoparticles under mild aqueous conditions is an unmet challenge. Genetically modified tobacco mosaic virus (TMV) can serve as a preferential precursor adsorption and growth sites for the controlled synthesis of palladium (Pd) nanoparticles with high catalytic activity. Here we describe detailed methods for the synthesis of Pd-TMV nanocomplexes as well as their integration into polymeric hydrogel microparticle platforms with controlled dimensions via a simple replica molding process. Such Pd-TMV-containing hydrogel particles may be useful in environmental remediation of toxic chemicals such as carcinogenic dichromate ions.

Integrated Methods to Manufacture Hydrogel Microparticles with High Protein Conjugation Capacity and Binding Kinetics via Viral Nanotemplate Display.

Genetically modified tobacco mosaic virus (TMV) can serve as a potent nanotemplate for high capacity protein conjugation through covalent coupling to its coat proteins with precise nanoscale spacing. TMV's own genomic RNA can also be exploited for orientationally controlled assembly onto various platforms with sequence and spatial selectivity via nucleic acid hybridization. Here we describe detailed methods for fabrication of hydrogel microparticles with capture DNA sequences, chemical activation and programming of TMV templates, TMV assembly with the microparticles and protein conjugation via bio-orthogonal click reactions.

High-throughput double emulsion-based microfluidic production of hydrogel microspheres with tunable chemical functionalities toward biomolecular conjugation.

Chemically functional hydrogel microspheres hold significant potential in a range of applications including biosensing, drug delivery, and tissue engineering due to their high degree of flexibility in imparting a range of functions. In this work, we present a simple, efficient, and high-throughput capillary microfluidic approach for controlled fabrication of monodisperse and chemically functional hydrogel microspheres via formation of double emulsion drops with an ultra-thin oil shell as a sacrificial template. This method utilizes spontaneous dewetting of the oil phase upon polymerization and transfer into aqueous solution, resulting in poly(ethylene glycol) (PEG)-based microspheres containing primary amines (chitosan, CS) or carboxylates (acrylic acid, AA) for chemical functionality. Simple fluorescent labelling of the as-prepared microspheres shows the presence of abundant, uniformly distributed and readily tunable functional groups throughout the microspheres. Furthermore, we show the utility of chitosan's primary amine as an efficient conjugation handle at physiological pH due to its low pKa by direct comparison with other primary amines. We also report the utility of these microspheres in biomolecular conjugation using model fluorescent proteins, R-phycoerythrin (R-PE) and green fluorescent protein (GFPuv), via tetrazine-trans-cyclooctene (Tz-TCO) ligation for CS-PEG microspheres and carbodiimide chemistry for AA-PEG microspheres, respectively. The results show rapid coupling of R-PE with the microspheres' functional groups with minimal non-specific adsorption. In-depth protein conjugation kinetics studies with our microspheres highlight the differences in reaction and diffusion of R-PE with CS-PEG and AA-PEG microspheres. Finally, we demonstrate orthogonal one-pot protein conjugation of R-PE and GFPuv with CS-PEG and AA-PEG microspheres via simple size-based encoding. Combined, these results represent a significant advancement in the rapid and reliable fabrication of monodisperse and chemically functional hydrogel microspheres with tunable properties.

Improved Protein Conjugation with Uniform, Macroporous Poly(acrylamide-co-acrylic acid) Hydrogel Microspheres via EDC/NHS Chemistry.

We demonstrate a robust and tunable micromolding method to fabricate chemically functional poly(acrylamide-co-acrylic acid) (p(AAm-co-AA)) hydrogel microspheres with uniform dimensions and controlled porous network structures for rapid biomacromolecular conjugation. Specifically, p(AAm-co-AA) microspheres with abundant carboxylate functional groups are fabricated via surface-tension-induced droplet formation in patterned poly(dimethylsiloxane) molds and photoinduced radical polymerization. To demonstrate the chemical functionality, we enlisted rapid EDC/NHS (1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)) chemistry for fluorescent labeling of the microspheres with small-molecule dye fluorescein glycine amide. Epifluorescence imaging results illustrate the uniform incorporation of carboxylate groups within the microspheres and rapid conjugation kinetics. Furthermore, protein conjugation results using red fluorescent protein R-phycoerythrin demonstrate the highly porous nature of the microspheres as well as the utility of the microspheres and the EDC/NHS scheme for facile biomacromolecular conjugation. Combined, these results illustrate the significant potential for our fabrication-conjugation strategy in the development of biofunctionalized polymeric hydrogel microparticles toward rapid biosensing, bioprocess monitoring, and biodiagnostics.

Integrated fabrication-conjugation methods for polymeric and hybrid microparticles for programmable drug delivery and biosensing applications.

Functionalized polymeric microparticles possess significant potential for controlled drug delivery and biosensing applications, yet current fabrication techniques face challenges in simple and scalable fabrication and biofunctionalization. For programmable manufacture of biofunctional microparticles in a simple manner, we have developed robust micromolding methods combined with biopolymeric conjugation handles and bioorthogonal click reactions. In this focused minireview, we present detailed methods for our integrated approaches for fabrication of microparticles with controlled 2D and 3D shapes and dimensions toward controlled release, and for biomacromolecular conjugation via strain promoted alkyne-azide cycloaddition (SPAAC) and tetrazine-trans-cyclooctene (Tz-TCO) ligation reactions utilizing a potent aminopolysaccharide chitosan as an efficient conjugation handle. We believe that the fabrication-conjugation methods reported here from a range of our recent reports illustrate the simple, robust and readily reproducible nature of our approaches to creating multifaceted microparticles in a programmable, cost-efficient and scalable manner toward a wide range of medical and biotechnological application areas.

Porosity-Tuned Chitosan-Polyacrylamide Hydrogel Microspheres for Improved Protein Conjugation.

We report a robust method to manufacture polyacrylamide-based functional hydrogel microspheres with readily tunable macroporous structures by utilizing a simple micromolding-based technique. Specifically, surface tension-induced droplet formation of aqueous solutions of chitosan and acrylamide in 2D-shaped micromolds followed by photoinduced polymerization leads to monodisperse microspheres. Pore sizes of the microspheres can be readily tuned by simple addition of inert long-chain poly(ethylene glycol) porogen at low content in the prepolymer solution. The as-prepared chitosan-polyacrylamide microspheres exhibit chemical functionality through chitosan's primary amines, rapid protein conjugation with selective tetrazine-trans-cyclooctene reaction, and nonfouling property. Combined with the potential to create anisotropic network structures, we envision that our simple fabrication-conjugation method would offer a potent route to manufacture a variety of biofunctionalized hydrogel microentities.

Shape-Encoded Chitosan-Polyacrylamide Hybrid Hydrogel Microparticles with Controlled Macroporous Structures via Replica Molding for Programmable Biomacromolecular Conjugation.

Polymeric hydrogel microparticle-based suspension arrays with shape-based encoding offer powerful alternatives to planar and bead-based arrays toward high throughput biosensing and medical diagnostics. We report a simple and robust micromolding technique for polyacrylamide- (PAAm-) based biopolymeric-synthetic hybrid microparticles with controlled 2D shapes containing a potent aminopolysaccharide chitosan as an efficient conjugation handle uniformly incorporated in PAAm matrix. A postfabrication conjugation approach utilizing amine-reactive chemistries on the chitosan shows stable incorporation and retained chemical reactivity of chitosan, readily tunable macroporous structures via simple addition of low content long-chain PEG porogens for improved conjugation capacity and kinetics, and one-pot biomacromolecular assembly via bioorthogonal click reactions with minimal nonspecific binding. We believe that the integrated fabrication-conjugation approach reported here could offer promising routes to programmable manufacture of hydrogel microparticle-based biomacromolecular conjugation and biofunctionalization platforms for a large range of applications.

Controlled Fabrication of Microparticles with Complex 3D Geometries by Tunable Interfacial Deformation of Confined Polymeric Fluids in 2D Micromolds.

Polymeric microparticles with complex shapes have attracted substantial attention in many application areas because particle shape is a critical parameter to impart programmable functionalities. The formation of specific three-dimensional (3D) microstructures in a simple, scalable, and controllable manner is difficult. Here, we report the controlled fabrication of microparticles with complex 3D shapes based on the simple tuning of mold swelling and capillarity. Specifically, a photocurable solution loaded in micromolds is spatially deformed into complex shapes depending on the degree of molding swelling and capillarity, thereby producing polymeric microparticles with controlled 3D shapes upon photopolymerization. The results show that highly uniform microparticles with controlled two-dimensional (2D) and 3D shapes were fabricated from identical 2D micromolds via the simple tuning of the wetting fluids. This technique can be extended to produce highly complex microarchitectures with controlled 3D geometric domains via 2D mold designs. Finally, multicompartment microparticles with independently controlled 3D shapes for each compartment are produced by a simple combination of fabrication sequences. We envision that this strategy of producing 3D microarchitectures from easily designed simple micromolds could provide a path to new materials and new properties.

Controlled fabrication of multicompartmental polymeric microparticles by sequential micromolding via surface-tension-induced droplet formation.

Polymeric multicompartmental microparticles have significant potential in many applications due to the capability to hold various functions in discrete domains within a single particle. Despite recent progress in microfluidic techniques, simple and scalable fabrication methods for multicompartmental particles remain challenging. This study reports a simple sequential micromolding method to produce monodisperse multicompartmental particles with precisely controllable size, shape, and compartmentalization. Specifically, our fabrication procedure involves sequential formation of primary and secondary compartments in micromolds via surface-tension-induced droplet formation coupled with simple photopolymerization. Results show that monodisperse bicompartmental particles with precisely controllable size, shape, and chemistry can be readily fabricated without sophisticated control or equipment. This technique is then extended to produce multicompartmental particles with controllable number of compartments and their size ratios through simple design of mold geometry. Also, core-shell particles with controlled number of cores for primary compartments can be readily produced by simple tuning of wettability. Finally, we demonstrate that the as-prepared multicompartmental particles can exhibit controlled release of multiple payloads based on design of particle compositions. Combined, these results illustrate a simple, robust, and scalable fabrication of highly monodisperse and complex multicompartmental particles in a controlled manner based on sequential micromolding.

An integrated approach for enhanced protein conjugation and capture with viral nanotemplates and hydrogel microparticle platforms via rapid bioorthogonal reactions.

We demonstrate significantly enhanced protein conjugation and target protein capture capacity by exploiting tobacco mosaic virus (TMV) templates assembled with hydrogel microparticles. Protein conjugation results with a red fluorescent protein R-Phycoerythrin (R-PE) show significantly enhanced protein conjugation capacity of TMV-assembled particles (TMV-particles) compared to planar substrates or hydrogel microparticles. In-depth examination of protein conjugation kinetics via tetrazine (Tz)-trans-cyclooctene (TCO) cycloaddition and strain-promoted alkyne-azide cycloaddition (SPAAC) reaction demonstrates that TMV-particles provide a less hindered environment for protein conjugation. Target protein capture results using an anti-R-PE antibody (R-Ab)-R-PE pair also show substantially improved capture capacity of R-Ab conjugated TMV-particles over R-Ab conjugated hydrogel microparticles. We further demonstrate readily controlled protein and antibody conjugation capacity by simply varying TMV concentrations, which show negligible negative impact of densely assembled TMVs on protein conjugation and capture capacity. Combined, these results illustrate a facile postfabrication protein conjugation approach with TMV templates assembled onto hydrogel microparticles for improved and controlled protein conjugation and sensing platforms. We anticipate that our approach can be readily applied to various protein sensing applications.

Facile strategy for protein conjugation with chitosan-poly(ethylene glycol) hybrid microparticle platforms via strain-promoted alkyne-azide cycloaddition (SPAAC) reaction.

We demonstrate a facile fabrication-conjugation scheme for protein-conjugated biosensing platforms. Specifically, we utilize a chitosan-poly(ethylene glycol) hybrid system to fabricate highly uniform and chemically reactive microparticle platforms via simple replica molding. Strain-promoted alkyne-azide cycloaddition (SPAAC) reaction between azide-modified proteins and microparticles activated with strain-promoted cyclooctynes allows tunable protein conjugation under mild reaction conditions. Upon conjugation of a model red fluorescent protein, fluorescence and confocal micrographs show selective protein conjugation near the particle surfaces as well as long-term stability of the conjugation scheme. Fluorescence and AFM results upon conjugation with varying protein concentrations indicate controllable protein conjugation. Examination of protein-particle conjugation kinetics shows multiple reaction regimes; rapid initial, intermediate, and steady final stage. Lastly, we demonstrate antibody conjugation with the particles and selective and rapid target protein capture with antibody-conjugated particles. Combined, these results illustrate a facile fabrication-conjugation scheme for robust protein-conjugated platforms that can be readily enlisted in various protein sensing applications.

A facile synthesis-fabrication strategy for integration of catalytically active viral-palladium nanostructures into polymeric hydrogel microparticles via replica molding.

The synthesis of small, uniform, well-dispersed and active Pd nanocatalysts under mild conditions in a predictable and controlled manner is an unmet challenge. Viral nanomaterials are attractive biotemplates for the controlled synthesis of nanoparticles due to their well-defined and monodisperse structure along with abundant surface functionalities. Here, we demonstrate spontaneous formation of small (1-2 nm), uniform and highly crystalline palladium (Pd) nanoparticles along genetically modified tobacco mosaic virus (TMV1cys) biotemplates without external reducing agents. The ratio between TMV and Pd precursor plays an important role in the exclusive formation of well-dispersed Pd nanoparticles along TMV biotemplates. The as-prepared Pd-TMV complexes are then integrated into the poly(ethylene glycol) (PEG)-based microparticles via replica molding (RM) technique in a simple, robust and highly reproducible manner. High catalytic activity, recyclability and stability of the hybrid Pd-TMV-PEG microparticles are further demonstrated through dichromate reduction as a model reaction. Taken together, these findings demonstrate a significant step toward simple, robust, and scalable synthesis and fabrication of efficient biotemplate-supported Pd nanocatalysts in readily deployable polymeric scaffolds with high capacity in a controlled manner.

Biologically inspired strategy for programmed assembly of viral building blocks with controlled dimensions.

Facile fabrication of building blocks with precisely controlled dimensions is imperative in the development of functional devices and materials. We demonstrate the assembly of nanoscale viral building blocks of controlled lengths using a biologically motivated strategy. To achieve this we exploit the simple self-assembly mechanism of Tobacco mosaic virus (TMV), whose length is solely governed by the length of its genomic mRNA. We synthesize viral mRNA of desired lengths using simple molecular biology techniques, and in vitro assemble the mRNA with viral coat proteins to yield viral building blocks of controlled lengths. The results indicate that the assembly of the viral building blocks is consistent and reproducible, and can be readily extended to assemble building blocks with genetically modified coat proteins (TMV1cys). Additionally, we confirm the potential utility of the TMV1cys viral building blocks with controlled dimensions via covalent and quantitative conjugation of fluorescent markers. We envision that our biologically inspired assembly strategy to design and construct viral building blocks of controlled dimensions could be employed to fabricate well-controlled nanoarchitectures and hybrid nanomaterials for a wide variety of applications including nanoelectronics and nanocatalysis.

Fabrication of chitosan-poly(ethylene glycol) hybrid hydrogel microparticles via replica molding and its application toward facile conjugation of biomolecules.

We demonstrate a facile scheme to fabricate nonspherical chitosan-poly(ethylene glycol) (PEG) microparticle platforms for conjugation of biomolecules with high surface density. Specifically, we show that PEG microparticles containing short chitosan oligomers are readily fabricated via replica molding (RM). Fluorescence and FTIR microscopy results illustrate that these chitosan moieties are incorporated with PEG networks in a stable manner while retaining chemical reactivity toward amine-reactive chemistries. The chitosan-PEG particles are then conjugated with single-stranded (ss) DNAs via Cu-free click chemistry. Fluorescence and confocal microscopy results show facile conjugation of biomolecules with the chitosan-PEG particles under mild conditions with high selectivity. These ssDNA-conjugated chitosan-PEG particles are then enlisted to assemble tobacco mosaic virus (TMV) via nucleic acid hybridization as an example of orientationally controlled conjugation of supramolecular targets. Results clearly show controllable TMV assembly with high surface density, indicating high surface DNA density on the particles. Combined, these results demonstrate a facile fabrication-conjugation scheme for robust biomolecular conjugation or assembly platforms. We expect that our approach can be enlisted in a wide array of biomolecular targets and applications.