No abstract

Large-scale nanoarrays of single biomolecules enable high-throughput assays while unmasking the underlying heterogeneity within ensemble populations. Until recently, creating such grids which combine the advantages of microarrays and single-molecule experiments (SMEs) has been particularly challenging due to the mismatch between the size of these molecules and the resolution of top-down fabrication techniques. DNA origami placement (DOP) combines two powerful techniques to address this issue: (i) DNA origami, which provides a ∼100 nm self-assembled template for singlemolecule organization with 5 nm resolution and (ii) top-down lithography, which patterns these DNA nanostructures, transforming them into functional nanodevices via large-scale integration with arbitrary substrates. Presently, this technique relies on state-of-the-art infrastructure and highly trained personnel, making it prohibitively expensive for researchers. Here, we introduce a cleanroom-free, $1 benchtop technique to create meso-to-macro-scale DNA origami nanoarrays using self-assembled colloidal nanoparticles, thereby circumventing the need for top-down fabrication. We report a maximum yield of 74%, 2-fold higher than the statistical limit of 37% imposed on non-specific molecular loading alternatives. Furthermore, we provide a proof-of-principle for the ability of this nanoarray platform to transform traditionally low-throughput, stochastic, single-molecule assays into high-throughput, deterministic ones, without compromising data quality. Our approach has the potential to democratize single-molecule nanoarrays and demonstrates their utility as a tool for biophysical assays and diagnostics.

3D-printed parts in PEEK for flow chemistry and continuous processing produced using fused filament fabrication.

Biofabrication methods capable of generating complex, three-dimensional, cell-laden hydrogel geometries are often challenging technologies to implement in the clinic and scaled manufacturing processes. Hydrogel injection molding capitalizes on the reproducibility, efficiency, and scalability of the injection molding process, and we adapt this technique to biofabrication using a library of natural and synthetic hydrogels with varied crosslinking chemistries and kinetics. We use computational modeling to evaluate hydrogel library fluid dynamics within the injection molds in order to predict molding feasibility and cytocompatibility. We evaluate the reproducibility of hydrogel construct molding and extraction and establish criteria for the selection of hydrogels suitable for injection molding. We demonstrate that hydrogel injection molding is capable of generating complex three-dimensional cell-laden construct geometries using diverse hydrogel materials and that this platform is compatible with primary human islet encapsulation. These results highlight the versatility and feasibility of hydrogel injection molding as a biofabrication technique with potential applications in the clinic and biomanufacturing.

Large-scale nanoarrays of single biomolecules enable high-throughput assays while unmasking the underlying heterogeneity within ensemble populations. Until recently, creating such grids which combine the unique advantages of microarrays and single-molecule experiments (SMEs) has been particularly challenging due to the mismatch between the size of these molecules and the resolution of top-down fabrication techniques. DNA Origami Placement (DOP) combines two powerful techniques to address this issue: (i) DNA origami, which provides a 100-nm self-assembled template for single-molecule organization with 5 nm resolution, and (ii) top-down lithography, which patterns these DNA nanostructures, transforming them into functional nanodevices via large-scale integration with arbitrary substrates. Presently, this technique relies on state-of-the-art infrastructure and highly-trained personnel, making it prohibitively expensive for researchers. Here, we introduce a bench-top technique to create meso-to-macro-scale DNA origami nanoarrays using self-assembled colloidal nanoparticles, thereby circumventing the need for top-down fabrication. We report a maximum yield of 74%, two-fold higher than the statistical limit of 37% imposed on non-specific molecular loading alternatives. Furthermore, we provide a proof-of-principle for the ability of this nanoarray platform to transform traditionally low-throughput, stochastic, single-molecule assays into high-throughput, deterministic ones, without compromising data quality. Our approach has the potential to democratize single-molecule nanoarrays and demonstrates their utility as a tool for biophysical assays and diagnostics.

Hydrogel injection molding is a biofabrication method that is useful for the rapid generation of complex cell‐laden hydrogel geometries, with potential utility in biomanufacturing products for tissue engineering applications. Hydrogel injection molding requires that hydrogel polymers have sufficiently delayed crosslinking times to enable injection and molding prior to gelation. In this work, we explore the feasibility of injection molding synthetic poly(ethylene) glycol (PEG)‐based hydrogels functionalized with strain promoted azide‐alkyne cycloaddition click chemistry functional groups. We evaluate the mechanical properties of a PEG‐based hydrogel library, including time to gelation and successful generation of complex geometries via injection molding. We evaluate the binding and retention of adhesive ligand RGD within the library matrices and characterize the viability and function of encapsulated cells. This work demonstrates the feasibility of injection molding synthetic PEG‐based hydrogels for tissue engineering applications, with potential utility in the clinic and biomanufacturing.

Biofabrication methods capable of generating complex, three-dimensional, cell-laden hydrogel geometries are often challenging technologies to implement in the clinic and scaled manufacturing processes. Hydrogel injection molding capitalizes on the reproducibility, efficiency, and scalability of the injection molding process, and we adapt this technique to biofabrication using a library of natural and synthetic hydrogels with varied crosslinking chemistries and kinetics. We use computational modeling to evaluate hydrogel library fluid dynamics within the injection molds in order to predict molding feasibility and cytocompatibility. We evaluate the reproducibility of hydrogel construct molding and extraction and establish criteria for the selection of hydrogels suitable for injection molding. We demonstrate that hydrogel injection molding is capable of generating complex three-dimensional cell-laden construct geometries using diverse hydrogel materials and that this platform is compatible with primary human islet encapsulation. We envision the application of this technique with primary cells via an off-the-shelf clinical kit that can be implemented at the patient bedside by a clinician or integrated with scaled, high-throughput automated cell manufacturing processes to generate encapsulated cell-based therapeutic products.Graphical Abstract