3D multi-modal shell for electrical and chemical monitoring and simulation

Abstract

Self-folding biocompatible shell microfluidic arrays (SMFAs) and methods for creating and using SMFAs. A SMFA configured as disclosed herein can include gradient or multilayer differentially crosslinked self-folding film layers, at least one leaflet; and at least one hollow microfluidic channel, where the at least one hollow microfluidic channel provides connection to at least one chemical source or sink connected to the at least one hollow microfluidic channel.

Description

3D MULTI-MODAL SHELL FOR ELECTRICAL AND CHEMICAL MONITORING AND SIMULATIONCROSS-REFERENCE

[0001] This application claims priority to U.S. provisional patent application no. 63 / 712,163, filed October 25, 2024, the contents of which are incorporated herein in their entirety.GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under grant numbers EFMA-2318093 and CBET-2348680 awarded by the United States National Science Foundation (NSF). The government has certain rights in the invention.BACKGROUND1. Technical Field

[0003] The present disclosure relates to a three dimensional (3D) multi-modal shell for electrical and chemical monitoring and simulation, and more specifically to customizable spherical microarrays of channels and pores for 3D spatiotemporal chemistry.2. Introduction

[0004] Microphysiological systems (MPS), such as organoids, are being explored as an animal alternative for disease modelling and drug development. However, interfacing with three-dimensional millimeter and submillimeter biomaterial is limited. Companies and labs often resort to the existing 2D platforms for the two-way interaction with the organoids. Thus, there exists a a need for 3D interfacing with the organoids. Given the availability of such 3D devices, companies and labs working on drug development and toxicity studies may soon move from 2D platforms to high-throughput 3D interrogation platforms.SUMMARY

[0005] Additional features and advantages of the disclosure will be set forth in the description that follows, and in part will be understood from the description, or can be learned by practice of the herein disclosed principles. The features and advantages of the disclosure can be realized and obtained by means of the instruments and combinations particularly pointed out in theappended claims. These and other features of the disclosure will become more fully apparent from the following description and appended claims, or can be learned by the practice of the principles set forth herein.

[0006] Disclosed are systems, methods, and non-transitory computer-readable storage media which provide a technical solution to the technical problem described. A self-folding biocompatible shell microfluidic array (SMFA), configured to perform concepts disclosed herein can include: gradient or multilayer differentially crosslinked self-folding film layers; at least one leaflet; and at least one hollow microfluidic channel, wherein the at least one hollow microfluidic channel provides connection to at least one chemical source or sink connected to the at least one hollow microfluidic channel.

[0007] An automated shell microfluidic array (SMFA) multimodal shell, configured to perform concepts disclosed herein can include: gradient or multilayer differentially crosslinked selffolding film layers; at least one leaflet; and at least one hollow microfluidic channel, wherein the at least one hollow microfluidic channel provides connection to at least one chemical source or sink connected to the at least one hollow microfluidic channel; and wherein the at least one microfluidic channel are configured to provide feedback between (1) a macrointerface and (2) controllers connected to chemical sources and electrodes.

[0008] A multimodal self-folding biocompatible shell microfluidic array (SMFA) configured to perform concepts disclosed herein can include: gradient crosslinked self-folding film layers with embedded fillers; at least one hollow microfluidic channel; electrical wiring: and contact pads coupled to the electrical wiring.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 illustrates examples of images of CAD-generated mask designs used in our multilayer photolithographic patterning of the 3D shell microfluidic arrays (SMFAs);

[0010] FIG. 2 illustrates an example schematic of 3D self-folding SMFA fabrication;

[0011] FIG. 3 illustrates an example of the removal of a sacrificial filler;

[0012] FIG. 4 illustrates an example of the self-folding of SU8 channels and immersion time in acetone;

[0013] FIG. 5 illustrates an example of self-folding behavior with varying width of the differentially exposed fold region (wf);

[0014] FIG. 6 illustrates an example of 3D self-folding microfluidics with different shapes;

[0015] FIG. 7 illustrates an example fo self-folding synthetic tendrils with different twisting behaviors;

[0016] FIG. 8 illustrates an example schematic of the physical bonding methodology used for interfacing external channels with the SMFAs;

[0017] FIG. 9 illustrates an example schematic of a chemical bonding methodology used for interfacing external channels with SMFAs;

[0018] FIG. 10 illustrates an example of interfacing for perfusion;

[0019] FIG. 11 illustrates an example of bead placement in an SMFA for spatiotemporal chemical patterning;

[0020] FIG. 12 illustrates an example CFD study illustrating examples of transient and steadystate gradients and pulsed flow;

[0021] FIG. 13 illustrates an example comparison between experiments and simulations of a transient gradient generated in a gel bead using an SMFA;

[0022] FIG. 14 illustrates an example comparison between simulations and experiments of a steady-state gradient in a gel bead generated using an SMFA;

[0023] FIG. 15 illustrates examples of a spatiotemporally controlled chemical reaction inside a gel bead using an SMFA;

[0024] FIG. 16 illustrates an example of neural organoid (NO) visibility inside an SFMA;

[0025] FIG. 17 illustrates examples of schematics, fabrication, and operation of the SMFAs;

[0026] FIG. 18 illustrates an example of wafer scale fabrication and self-folding characteristics of SMFAs;

[0027] FIG. 19 illustrates an example of self-folding microfluidics with bioinspired shapes;

[0028] FIG. 20 illustrates an example of controlled 3D multi-chemical microfluidic dispensation through SMFAs;

[0029] FIG. 21 illustrates an example of CFD-assisted gradient pattering of gel beads using SMFAs;

[0030] FIG. 22 illustrates an example of multi-chemical localized spatial gradient of gel beads;

[0031] FIG. 23 illustrates examples of 3D microchemistry inside a gel bead by spatiotemporal release of reactants using SMFAs;

[0032] FIG. 24 illustrates an example of a biocompatibility assessment and selective straining of neural organoids (NOs);

[0033] FIG. 25 illustrates an example of Multimodal microelectrofluidic array (MEFA) fabrication process flow;

[0034] FIG. 26 illustrates an example of potential applications of MEFA;

[0035] FIG. 27 illustrates an example of a multimodal MEFA;

[0036] FIG. 28 illustrates examples of fluidic and electrical interfacing with a multimodal MEFA; and

[0037] FIG. 29 illustrates an example computer system.DETAILED DESCRIPTION

[0038] Various embodiments of the disclosure are described in detail below. While specific implementations are described, this is done for illustration purposes only. Other components and configurations may be used without parting from the spirit and scope of the disclosure.

[0039] Microscale patterning of both unreactive and reactive chemicals with 3D spatiotemporal control occurs in a wide range of natural and industrial processes, including self-organization, emergence, dynamical systems, embryogenesis, reaction-diffusion networks, and micro physiological systems. For example, 3D micropatterning of unreactive chemicals is vital for synthesizing patchy particles and complexes for self-assembly, colloidal science, and programmable matter. While two-sided Janus particles have advanced colloidal science, approaches to creating particles with more complex 3D patterns could significantly enhance the functionality of particles. The 3D microscale delivery and spatiotemporal patterning of reactive chemicals significantly influence reaction-diffusion networks, leading to unexpected pattern formation and emergence. Functional polymeric particles produced using microfluidic multiemulsion synthesis methods can facilitate chemical encapsulation and targeted drug delivery.

[0040] In biology, 3D spatiotemporal chemistry is abundant within single cells and subcellular structures. For instance, the precise localization of proteins and protein complexes plays a crucial role in bacterial cell division. Similarly, 3D biochemical patterns, such as morphogen gradients and spatially encoded transcriptional activators and inhibitors, are fundamental to embryogenesis, morphogenesis, and disease progression. Morphogen gradients in early embryonic development typically emerge at sub-millimeter scales in 3D and evolve over minutesto hours before being replaced by new gradients that guide further structural development.Replicating these dynamic biochemical patterns in controlled environments requires precise 3D microfluidic engineering approaches.

[0041] Conventional microfluidics, which include channels and sources on flat substrates, have been widely utilized to create chemical patterns such as gradients and arrays of diffusible sources. For example, some have used microfluidic networks to develop complex planar gradients of laminin, which control axon specification. Elsewhere, stacked microfluidic channels containing fluids of different oxygen concentrations can provide the oxygen gradient required for gut microbiome culture in an anaerobic intestine-on-a-chip. Studies using fluid patterning have also shed light on the role of morphogens such as Wingless-related Integration Site (WNT) in intestinal morphogenesis and neural tube development, as well as bone morphogenetic protein 4 (BMP4) gradients in inducing asymmetric cell fate and guiding neural cell growth. Moreover, in vitro studies using two-dimensional (2D) microfluidics have demonstrated that temporally regulated drug treatments can be more effective than constant-dose therapy. Despite significant advances, 2D microfluidic approaches still struggle to achieve precise 3D spatiotemporal control over micropatterning, microscale chemical reactions, and manipulation of the extracellular microenvironment, particularly in curved geometries.

[0042] With the emergence of the field of micro physiological systems focusing on organ-on-chip, 3D tissue engineering, and organoid biology, researchers have recently developed biomimetic microfluidic systems that can modulate and pattern the chemical microenvironment in 3D. For example, others have developed 3D-printed cube-shaped culture devices to create a 3D morphogen gradient of ectoderm and neuroectoderm lineages. Still others fabricated a tetrahedrally arranged, 3D microfluidic system to generate spatial gradients for combination antimicrobial susceptibility testing. Nevertheless, significant challenges still exist with 3D submillimeter chemical patterning, especially regarding ease of use, resolution, temporal control, scalability, and biocompatibility. Moreover, the cytoarchitecture and size of diverse organoids and assembloids are becoming increasingly complex as the field advances, demanding increasingly sophisticated micro instrumentation.

[0043] Here, we describe self-folding biocompatible shell microfluidic arrays (SMFAs) for spatiotemporal chemical patterning in a 3D microenvironment, thereby enabling precise chemical patterning and chemistry within 3D curved microenvironments. We utilizeconventional photolithography to create gradient crosslinked self-folding SU8 films with embedded sacrificial fillers through wafer-scale batch fabrication. After fabrication, we dissolve the fillers to create hollow microfluidic channels with widths as small as 200 pm. Self-folding can generate a variety of curved microfluidic shapes, including cylinders, spirals, and spherical shells. The fabrication and assembly process is reproducible and tunable.

[0044] Focus is given to millimeter-sized shell structures composed of self-folding leaflets, chosen for their spherical geometry, symmetry, and suitability for patterning spherical objects. We use a combination of computational fluid dynamics (CFD) simulations and physical experiments to demonstrate the broad applicability of our SMFAs. We demonstrate that we can distribute channels and pores as needed around any 3D microcavity or object, such as a spherical particle, gel bead, or organoid. The SMFAs can be designed such that portions adhere to the wafer substrates on which they are fabricated for facile packaging and integration with other modules. We are thus able to connect them to programmable external flow controls with polydimethylsiloxane (PDMS) inlets and outlets (see Fig. 17a).

[0045] We highlight the broad utility of SMFAs by demonstrating (a) transient and steady-state chemical gradients, (b) multi-chemical patterning, and (c) multi -reactant chemical reactions within millimeter-sized gel beads (see Fig. 17b-e). We also demonstrate the biocompatibility of our SMFAs and their capabilities in microphysiological systems by spatiotemporally patterning induced pluripotent stem cell (iPSC)-derived neural organoids (NOs). These studies establish the capabilities and widespread applicability of the SMFAs platform.

[0046] Concept and fabrication of self-folding SMFAs

[0047] SMFAs consist of self-folding leaflets integrated with inlet and outlet microfluidic channels, interfaced with pumps using irreversible Polydimethylsiloxane (PDMS) bonding. The SMFAs effectively distribute 3D chemical sources (CS) in a spherical geometry around objects such as gel beads and NOs. Chemicals can be dispensed through the inlets in situ with temporal control using syringe pumps and microfluidic digital controllers.

[0048] The self-folding shell incorporates several distinct and essential elements. First, each leaflet is composed of a differentially UV-crosslinked photoresist (SU8) trilayer, whose extent of crosslinking is varied such that it bends with a pre-designed curvature upon release from the substrate. Bending is driven by a mismatch in the volumetric change of the different layers of the trilayer in water or cell media after an acetone treatment due to their differential crosslinking. Wecan tune the extent of bending of the trilayer to wrap around spherical objects by varying the thickness of the layers and the extent of UV exposure of each layer. Second, we incorporate sacrificial fillers between the bending trilayer to create folding microfluidic channels. Lastly, we integrate the microfluidic channels and bending leaflets with PDMS inlets and outlets, connecting them to external microfluidic controllers for turnkey operation (see Fig. 17f and g).

[0049] The fabrication process begins with six computer-aided design (CAD) photomasks featuring alignment marks, allowing for precise overlay during UV lithography (see Fig. 1). The first photomask defines the thermally evaporated germanium (Ge) sacrificial layer that is dissolved at the end of the patterning process to release selected portions of the shell leaflets (see Fig. 2). We used three photomasks to define the first, second, and third SU8 layers of the selffolding trilayer and microfluidic channel. The fifth and sixth photomasks define the sacrificial filler and the second exposure of the third SU8 layer (double exposure to tune bending). The folding region of the third SU8 layer is single-exposed (300 mJ / cm2) compared to the other areas, which are exposed twice (cumulative 500 mJ / cm2), creating a bend-inducing gradient selectively at the folding region. We experimented with several sacrificial materials, including readily dissolvable metals such as copper, unexposed SU8 followed by dissolution in developer, and various photoresists. We found that the photoresist SPR220 works best as a sacrificial filler due to its ability to be photopattemed and dissolve in SU8 developer, without interfering with the self-folding capability of the trilayer devices. After the lithography patterning process, we dissolve this sacrificial filler to create hollow channels inside the SU8 trilayer (see Figs. 2 and 3). The underlying Ge sacrificial layer is then dissolved, releasing the SU8 leaflets and forming selffolded microfluidics (see Fig. 17g). The fabrication process for SMFAs is wafer-scale, offering cost-effective batch processing with reproducible bending (see Fig. 18a-b).

[0050] Design considerations for self-folding microfluidics

[0051] While we primarily focus on SMFA shapes in this study, our fabrication protocol is adaptable for designing curved and folded microfluidic systems with various bioinspired shapes. Nature presents numerous curved and folded 3D fluidic designs, including leaves, tendrils, and pulmonary capillaries. Creepers grab on to support by forming helical tendrils; leaves protect themselves from photodamage and transpiration in arid environments by reducing the exposed area by self-rolling; and pulmonary capillaries wrap around the spherical alveoli for effective gas exchange. Inspired by naturally occurring designs, we fabricated various self-foldingmicrofluidic shapes. To design such shapes, we note that many variables can influence the bending of SU8 fluidic channels to tune the extent of self-folding, including the thickness and lateral dimensions of the differentially crosslinked SU8 regions, the UV exposure energy gradient, and duration of acetone treatment. In this study, we identified the duration of acetone treatment required (1 hour) for localized bending at the differentially crosslinked region, with a fixed value of the SMFA thickness (~35 pm) and differential exposure (300 / 500 mJ / cm2) (see Fig. 4). Then, we tuned the folding behavior by varying the width of the differentially exposed folding region of the trilayer (w / ) (see Figs. 5 and 18). We observed a relatively monotonic increase in bending angle 9 with respect to w (see Fig. 18c), which enabled us to fabricate selffolding channels with varying curvatures and shapes.

[0052] To demonstrate the versatility of our self-folding method, we created 3D microfluidic devices with different shapes (see Figs. 6, 18, and 19). We fabricated helical microfluidic structures that mimic tendrils by creating differentially crosslinked trilayer fold regions angled in the longitudinal direction (see Figs. 18d and 19a). By tuning w and the distance between adjacent fold regions, we created synthetic tendrils of different twisting behaviors and radii of curvatures (see Figs. 7, 18d, and 19a). Self-folding cylindrical microfluidic shapes with multiple channels akin to a rolled leaf were also fabricated, as shown in Fig. 19b. Additionally, selffolding SMFAs with varying curvature, pore layouts, and multiple channels were created (see Fig. 19c, d).

[0053] Controlled perfusion and patterning through CSs in SMFAs

[0054] To connect the SMFAs to external fluidic instrumentation (e.g., syringe pumps, flow controllers), macro-to-micro inlets and outlets were created using a PDMS slab following conventional microfluidic protocols (see Fig. 8-10). Briefly, the PDMS was bonded to the SU8 using a plasma bonding process assisted by (3 -aminopropyl) triethoxysilane (APTES). We used a biopsy punch to define inlets and outlets for connecting tubing. Typically, we perfused the channels in 3D self-folding SMFAs using multi-channel syringe pumps at flow rates of 1-20 pL / min. We visualized the perfusion of one-, three-, and five-channel SMFAs using dyes and observed reliable fluid flow without leakage (see Fig. 20a-c). These studies demonstrated that multiple channels and CSs can be engineered into SMFAs, and temporal control can be achieved through the use of appropriate macro-scale digital controllers and by manipulating flow rates (see Fig. 20d).

[0055] The controlled spatiotemporal placement of CSs within SMFAs enables the creation of complex chemical patterns and gradients with broad applicability in micropatterning and the biomedical sciences. While such patterns have traditionally been applied in 2D systems, SMFAs extend this capability to 3D environments, particularly for studying spherical structures. To study gradient generation inside spherical beads, a gel bead approximately 2 mm in diameter was first placed in the shell while the SMFA was folding (Fig. 11). Since self-folding occurs over 24 hrs, the SMFA can wrap around the object as the folding progresses. We generated transient and steady-state gradients within agarose beads by releasing fluorescent dyes (rhodamine 6G and fluorescein) from specific CSs in the SMFA. The transient and steady-state gradient profiles were designed and simulated in COMSOL 6.2 using a diffusion constant of (D = 4 x 1010m2s *) and a laminar flow rate of 5 pL / min. To study the transient gradient, we perfused 10 mol / m3rhodamine 6G dye (diluted in DI H2O) through the agarose hydrogel bead (see Fig. 21a) using one of the three leaflets of the SMFAs. Over 20 minutes, we observed that the advancing fluorescence front diffusing through the bead is in good agreement with the simulation results (see Fig. 21a). We note that by manipulating the flow rates through different pores and diffusion constants, a variety of transient gradients can be formed (see Figs. 12-14). We also designed a steady state gradient across a gel bead using CFD simulations by introducing two solutions containing different target chemicals through diametrically opposite inlets surrounding the bead, as shown in Fig. 21b. We validated the devised gradient generation using the fabricated SMFAs by introducing water with fluorescein and rhodamine 6G through two pores of the SMFA (see Fig. 21b).

[0056] We conducted experiments demonstrating the SMFAs' ability to multichemically micropattern agarose gel beads. Using a three-leaflet SMFA, we perfused two or three different chemicals (blue, red, and green dyes) through CS1, CS2, and CS3 at a flow rate of 5 pL / min. The dyes diffused within the gel bead from different directions to create multicolor patterns, which can be manipulated by controlling flow rates, pulsed flow, and reactivity (see Fig. 22 and Figs. 12 and 14).

[0057] 3D spatiotemporal chemistry using SMFAs

[0058] We demonstrate chemical reactions with spatiotemporal control within gel beads. By combining flow and transport of species through porous media, we simulated the transport of reacting chemicals through the bead. We dispensed two reactive chemical solutions, sodiumcarbonate (Na2COs) in water and magnesium sulfate (MgSCL) in water, through spatially separated CSs and studied the double displacement reaction that forms magnesium carbonate (MgCCh) precipitates.

[0059] The reactants diffused through the porous medium of the gel bead. At the points of contact where the diffusion fronts of dissolved ions met, they produced an insoluble MgCCh with a distinctive white color that can be visualized within the bead (see Fig. 15 and 23). To study the precipitation profile using the CFD model, we assumed that the precipitation rate of MgCCb is directly proportional to the product of the concentrations of Mg2+and CCh2'. The product of the concentrations obtained from CFD is plotted. The corresponding experimental results match the profiles, as shown in Fig. 23. Notably, SMFAs enable the localization of the chemical products in different regions within the bead by varying the locations of the CSs. For example, when one CS of MgSCU is used in the SMFA, then a single spheroidal MgCCb precipitate is produced, whereas with two CSs of MgSO4, a hemispherical-shaped MgCCh precipitate is produced within the bead (see Fig. 23). Due to the large number of potential design parameters of CSs, chemical reactants, and flow rates, a variety of other 3D localized chemical product patterns are possible. We anticipate that microfluidic systems can be adapted and scaled for use in vitro biological models to facilitate high-throughput testing of drug interactions, drug metabolism, and toxicity.

[0060] Self-folding SMFAs for 3D tissue engineering and organoid research

[0061] We envision that SMFAs can mimic and control the extracellular environment of in vitro cellular models with multiple basic and translational applications. We propose that more complex arrangements of spatiotemporal gradients and localized chemical reactions could replicate the spatial information encoded near a developing embryo, allowing for further insights into the fine-tuned processes that govern embryogenesis within microphy si ologi cal systems.

[0062] Although the materials used to fabricate SMFAs have been previously utilized in tissue engineering devices, we first verified their biocompatibility with NOs. Briefly, we cultured NOs from iPSCs using previously described protocols, which resulted in cells reaching approximately 1 mm in diameter. We placed them within the self-folding SMFAs and evaluated the biocompatibility of the SMFAs using Lactate Dehydrogenase (LDH)-Glo™ and resazurin assays. The release of LDH enzyme into the medium, following cell membrane damage, is a cytotoxicity marker. We monitored LDH release over 72 hrs across six time points (see Materials and Methods). We selected a 72-hour time point to align with our organoid mediachange schedule (every 2-3 days). The resazurin assay, performed at the 72-hour time point, assessed cellular metabolic activity by measuring the reduction of resazurin to resarufin, serving as an indicator of cell viability. Both assays showed no statistically significant differences between the control and experimental groups, confirming that the device is non-cytotoxic and biocompatible with neural cell cultures (see FIGs. 16 and 24a). Furthermore, we evaluated the viability by maintaining NOs within the shell for up to one week (see FIGs. 16 and 24b). The green stain indicates the presence of neurites between the organoid and the SMFA leaflet, demonstrating the active neurite outgrowth feature of healthy organoids, which highlights viability.

[0063] We demonstrated localized perfusion on living 1 mm NOs (Fig. 24c). To demonstrate the 3D spatiotemporal patterning of NOs, we used an SMFA with three channels (see schematic in Fig. 24c) and placed two NOs within the shell, each positioned near different leaflets. We perfused leaflet 1 with Hoechst 33342 trihydrochloride stain at a dilution of 1:2000 in media, and the bottom leaflet with unaltered media for 7 minutes at a flow rate of 5 pL / min. We performed fluorescence imaging after fixing the organoids while they were inside the SMFA. Significant fluorescence was observed only in the organoid closest to the leaflet perfused with Hoechst, as shown in Fig. 24c. We observed that NO1 appeared blue. In comparison, there was no significant fluorescence from NO2 (see Fig. 24c). This selective staining confirms the SMFA's ability to achieve spatial patterning, rather than allowing indiscriminate dye diffusion throughout the shell's interior. These results demonstrate the potential for precisely directing biochemicals to organoids in 3D space, whether targeting specific organoids or distinct regions within a larger organoid, thereby enabling controlled modulation of the microenvironment.

[0064] In summary, using both unreactive and reactive chemicals, we have demonstrated selffolding SMFAs for 3D spatiotemporal chemistry. Key features of SMFA fabrication include customization using CAD design, the precision and reproducibility of planar lithography, the scalability and cost efficiency of wafer-level processing with the capability of integration with other microelectronic or microelectromechanical systems (MEMS) components. Notably, unlike other approaches, such as buckling on pre-stretched substrates, our method eliminates the need for substrate transfer steps, which can limit manufacturability and integration with optical, magnetic, electrical imaging modules, and biosensors. Also, the SMFA is composed of a widely used and biocompatible photoresist, SU8. The extent and type of self-folding can be engineeredby varying the type and extent of crosslinking, and when combined with photopattemable sacrificial fillers, allow the creation of curved and folded microfluidic systems inspired by biological structures and anatomically relevant designs. Such curved microfluidics are widely observed in nature, such as in leaves, skin, villi, and the brain, and are challenging to create using other methods, including additive manufacturing, due to the need for support structures and the requirement for small features. Moreover, the presented method offers shape-changing capability, enabling the encapsulation of 3D objects. In this study, we self-folded several bioinspired microfluidic designs including synthetic tendrils, multi-source tubes, and sphereencapsulating shells and the approach is amenable to more complex designs by incorporating origami and kirigami design principles.

[0065] Apart from folding and curvature parameters, several variables can be tuned through integration with digital flow controllers and CAD designs of channels and pores, including the types, spatial locations, and temporal characteristics of CSs. In this regard, we have utilized CFD to guide our experiments and have demonstrated the ability to pattern and create gradients within gel beads with high 3D spatiotemporal precision. In the future, artificial intelligence (Al) approaches, inverse design, and optimization methods could significantly enhance the complexity and spatiotemporal characteristics of chemical patterns and reactions.

[0066] The biocompatibility of the SMFAs and the ability to tune the 3D spherical spatial localization of CSs, combined with the macro-to-microfluidic interface, can enable a variety of morphogen, biochemical, and extracellular signaling molecular patterns. Such gradients are essential for investigating important questions, such as the 3D spatiotemporal effects of quorum sensing and chemotaxis in bacteria, directional growth of slime molds towards food sources, and embryonic development. The system is valuable for investigating emergence, self-organization, and dissipative reactions in complex 3D geometries. For instance, it enables the controlled dispensing of chemical reactants used in the Belousov-Zhabotinsky reaction and oscillatory reaction-diffusion systems from the CSs in the SFMAs.

[0067] Additionally, the self-folding approach is highly versatile in terms of the materials used. While we have utilized SU8 in our study, gradient and UV crosslinking can also be applied to other photopattemable materials, such as gels, opening significant opportunities for recreating biomimetic and microphysiological systems, such as the embryonic sac or eggshell membranes. Such 3D fluidic environments can enable spatiotemporal differentiation and mimic key aspectsof embryonic development. Additionally, just as chemicals can be dispensed through chemical sources, they can also be retracted by applying negative pressure to the pores within the SMFAs, which could facilitate spatiotemporal microsampling of the microenvironments surrounding biological systems and organoids.

[0068] MATERIALS AND METHODS

[0069] Microfabrication of channels

[0070] A detailed process flow, including figures and masks, is shown and described below, beginning with FIG. 1. Briefly, we spin-coated a layer of ~2.7-pm-thick SI 827 photoresist (Kayaku, Westborough, MA) on a 3-inch silicon wafer (NanoSilicon, Inc., USA) at 3000 rpm to create mask patterns for the germanium (Ge) sacrificial layer. This positive photoresist was then exposed using an NXQ-4000 mask aligner with a mercury arc lamp (Neutronix-Quintel, Morgan Hill, CA) through a photomask (designed in Autodesk AutoCAD 2024) at an energy density of 150 mJ / cm2to selectively open the windows to the silicon underneath the polymer. We removed the exposed portion of the resist by treating it with 351 developer (diluted 1:5 with water, Kayaku, Westborough, MA) for one minute. Then, a 50-nm-thick Ge layer was deposited on the silicon wafer through the patterned SI 827 photoresist via thermal evaporation. We employed a lift-off process using acetone to define the Ge sacrificial layer, upon which the self-folding leaflets were fabricated through a trilayer lithography method.

[0071] The microfluidic channels comprise three SU8 layers that define the base, sidewalls, and top of the channels. We used the following parameters for the layers.

[0072] Layer 1 SU82005 (Spin speed: 3000 rpm; soft bake: 1 min, 3 mins, and 1 min at 65°C, 95°C, and 65°C, respectively; exposure energy density: 240 ml / cm2; post-exposure bake: 1 min, 3 mins, and 1 min at 65°C, 95°C, and 65°C, respectively, development duration: 1 min 30 sec).

[0073] Layer 2 SU82015 (Spin speed: 3000 rpm; soft bake: 1 min, 10 mins, and 1 min at 65°C, 95°C, and 65°C, respectively; exposure energy density: 500 mJ / cm2; post-exposure bake: 1 min, 10 mins, and 1 min at 65°C, 95°C, and 65°C, respectively, development duration: 2 min).

[0074] Layer 3 (Dual exposure): SU82015 (Spin speed: 3000 rpm; soft bake: 1 min, 10 mins, and 1 min at 65°C, 95°C, and 65°C, respectively; exposure energy density: 300 mJ / cm2(leaflets) and 500 mJ / cm2(the rest of the region); post-exposure bake (performed 24 hr after the exposure): 1 min, 10 mins, and 1 min at 65°C, 95°C, and 65°C, respectively, development duration: 7 days).

[0075] Between layers 2 and 3, we patterned a sacrificial filler for the channels using the following process parameters.

[0076] Sacrificial filler'. SPR220 (Spin speed: 1700 rpm; soft bake: 1 min at 115 °C; exposure energy density: 500 mJ / cm2; development duration: 2 min).

[0077] The seven-day development of the structure in SU-8 developer after Layer 3 fabrication ensured the complete removal of the sacrificial filler.

[0078] To release the leaflets from the Ge sacrificial layer for self-folding, we dissolved the layer by placing the wafer inside 6% hydrogen peroxide for 24 hrs. Following the complete dissolution of the Ge sacrificial layer, we immersed the wafers in acetone for 1 hr to precondition the folding of the SMFAs. The final step in the fabrication process is the induction of selffolding, which occurs through a solvent exchange process by immersing the acetone-treated structure (1 hour) in water. In some configurations the times can vary (e.g.,+ / - 10%), according to specific configuration and preference.

[0079] Interfacing with the pump

[0080] We bonded a polydimethylsiloxane (PDMS) slab with holes to the inlet / outlet patterns of the self-folded microfluidic devices, allowing for interfacing with external pressure sources (see Figs. 8 and 9). Briefly, we made the PDMS mold on a silicon wafer using SU82025 photoresist (Kayaku, Westborough, MA). We then poured a homogeneous mixture of Sylgard PDMS base and curing agent (Dow Silicones Corporation, Midland, MI) in a 10:1 mass ratio onto the SU8 mold and cured it at 90 °C for 10 minutes, followed by 120 °C for 50 minutes. The molded PDMS was removed and cut into the required size. Before bonding it to the self-folding microfluidics, we punched holes in these PDMS slabs where the microfluidic tubing had to be inserted.

[0081] We present two different bonding strategies to ensure fluidic sealing between SU8 and PDMS: chemical bonding using (3 -aminopropyl) tri ethoxy silane (APTES) or physical bonding using a double-sided adhesive. We primarily used the chemical bonding process derived from the following protocol. Briefly, we activated the face of the PDMS piece to be bonded using oxygen plasma (50 W for 1 minute). The activated PDMS and SU8 were treated with 5% APTES solution for 20 minutes and bonded by aligning the punched holes with the inlets / outlets on the self-folded microfluidic devices, then heating the assembly for 30 minutes at 80°C. Afteraccomplishing a chemical or physical bonding, we inserted microfluidic tubes into the punched holes. We secured them using superglue to prevent leakage from the insertion point.

[0082] Agarose bead synthesis

[0083] We generated agarose beads of uniform volume by diluting solid agarose to 1.5% w / w in water and stirring, then melting it in a microwave for 1 minute. We removed large bubbles by placing the agarose inside a vacuum chamber for 10 minutes while the gel cooled. Then, we cut a large piece of gel and submerged it in a vial of mineral oil. We heated the vial to 50 °C on a hot plate, causing the agarose piece to form a large bubble inside the oil. Using a 2 pL micropipette, we drew up 1 pL of the molten agarose in a 10 pL pipette tip. We then carefully ejected the contents submerged in a vial of room-temperature mineral oil. The agarose quickly solidifies into a spherical bead. We repeated this process until we had the desired number of beads for performing experiments. We washed the beads with DI water and stored any excess beads in a water-filled petri dish for further use.

[0084] Computational simulation of fluid delivery and gradient dynamics

[0085] We used COMSOL Multiphysics (COMSOL 6.2) to simulate the fluid delivery and gradient dynamics within our designed microfluidic shell, focusing on two scenarios: a transient gradient and a steady-state gradient. Both setups involved CSs placed to control flow distribution within a spherical hydrogel region. We modeled the hydrogel sphere as a homogeneous and isotropic porous zone with a constant porosity of c = 0.99. For the transient gradient, we set one CS with a concentration of 10 mmol / m3with a flow rate of 5 pL / min. For simulating the steadystate gradient, we set one CS with an inflow concentration of 10 mmol / m3, and the CSs on the diametrically opposite side had a concentration of 0 mmol / m3. We perfused both CSs at a flow rate of 5 pL / min. Using Rhodamine 6G as the diffusing molecule with a diffusion coefficient of D = 400 pm2 / s, the simulation demonstrated the establishment of both transient and steady-state gradients.

[0086] Differentiation of NOs from induced pluripotent stem cells

[0087] We differentiated NOs from the NIBSC-8 induced Pluripotent Stem Cell (iPSC) line (female origin, UK National Institute for Biological Standards and Control (NIBSC)). We confirmed that the iPSC line was mycoplasma-free and had a normal karyotype. We then performed the differentiation using a two-step protocol. Briefly, we cultured iPSCs in mTESR-Plus medium (STEMCELL TECHNOLOGIES) under standard conditions (5% O2, 5% CO2,37°C). Then, we induced neural differentiation with a serum-free, chemically defined neural induction medium (GIBCO, THERMO FISHER SCIENTIFIC) to generate neural progenitor cells (NPCs). For NO formation, we seeded 2 x 106NPCs per well into uncoated 6-well plates to form 3D aggregates under constant gyratory shaking (88 rpm, 19 mm orbit) at 37°C, 5% CO2, and 20% O2. After 48 hrs, we induced differentiation by switching to a serum-free, chemically defined differentiation medium (Neurobasal Plus medium) supplemented with lx B27-Plus supplement, 2% Glutamax (GIBCO, THERMO FISHER SCIENTIFIC), 10 ng / mL human recombinant Glial Cell-Derived Neurotrophic Factor (GDNF) (GEMINIBIO), 10 ng / mL human recombinant Brain-Derived Neurotrophic Factor (BDNF) (GEMINIBIO), and 1% Penicillin-Streptomycin (GIBCO, THERMO FISHER SCIENTIFIC). We replaced approximately 75% of the medium every 2-3 days.

[0088] Biocompatibility assays

[0089] We cultured eight-week-old NOs for 72 hrs in static conditions (no gyratory shaking) at 37°C and 5% CO2. The control group consisted of five wells, each containing ten organoids, while the experimental group consisted of five wells containing one SMFA and ten organoids. According to the manufacturer's protocol, we assessed cytotoxicity using the Lactate Dehydrogenase (LDH)-Glo™ Cytotoxicity Assay (Promega J2381). Briefly, we collected media samples at six time points: 1, 6, 12, 24, 48, and 72 hrs. At each time point, we mixed 5 pL of media from each well with an LDH storage buffer and stored at -20°C for later analysis. At the 72-hour time point, we transferred 50 pL of each sample to a 96-well luminescence plate and combined it with 50 pL of LDH detection reagent. The reaction proceeded for 30 minutes, after which we measured the relative luminescence units (RLU) using a Promega Glomax MultiDetection microplate reader.

[0090] We conducted a resazurin assay at the 72-hour time point to assess cell viability. After media collection, we added 0.1 mg / mL of resazurin to each well and incubated the plates for 3.5 hrs at 37°C. After incubation, we transferred 100 pL of media from each well to a 96-well plate for fluorescence-based analysis. We measured the fluorescence using an excitation wavelength of 530 / 25 nm and an emission wavelength of 590 / 35 nm.

[0091] Dye gradient staining of Nos

[0092] We placed a sterile cloning cylinder around the leaflets of the SMFA to contain the media and affixed to it the SU-8 wafer with uncured PDMS. We then autoclaved all plastic tubing andsterilized the entire setup by immersing it for 15 minutes in 100% isopropyl alcohol, followed by 15 minutes of drying and then washing with sterile Phosphate Buffered Saline (PBS) and basal KnockOut media. After placing the organoid in the leaflets, we perfused leaflet 1 with Hoechst 33342 trihydrochloride stain at a dilution of 1 :2000 and a rate of 5 pL / min for 7 minutes. We then processed the organoids for immunofluorescence microscopy.

[0093] Immunofluorescence

[0094] We fixed the NOs with 4% PFA for 45 min at room temperature, followed by three rinses with 1% Bovine Serum Albumin (BSA) in PBS, permeabilized and blocked for 1 hr in 10% normal goat serum, 1% BSA, and 0.15% saponin in PBS (blocking solution). We incubated the NOs at 4 °C for 24 h with antibodies against P-III-Tubulin and Glial Fibrillary Acidic Protein (GFAP) (Table 1) in a blocking solution. P-III-Tubulin and GFAP are key markers for early neuronal and astrocyte differentiation, respectively. We then washed the NOs with 1% BSA and 0.15% saponin in PBS three times and incubated them with secondary antibodies (Table 2) in a blocking solution at 4 °C for 24 hours. We washed the NOs three times with PBS containing 1% BSA and imaged them with a Nikon AZ100 microscope (Nikon, Tokyo, Japan).

[0095] Table 1. Primary antibodies used for immunofluorescence.Primary antibody Host Type Source / Cat No. DilutionP-Tubulin Class III Mouse Monoclonal Sigma T5076 1:1500GFAP Rabbit Polyclonal DAKO Z0332 1:400

[0096] Table 2. Secondary antibodies used for immunofluorescence.Secondary Antibody Host / Target Type Source / Cat No. DilutionAlexa Fluor 488 Goat / Mouse Polyclonal IgG Invitrogen A-21042 1:500Alexa Fluor 568 Goat / Rabbit Polyclonal IgG Invitrogen A-l 1011 1:500

[0097] An embodiment of the current invention is to combine 3D microfluidics and electrical stimulation / recording within a single spherical shell to precisely control, manipulate and monitor BOTH the chemical and electrical microenvironment of 3D cell culture and organoids.

[0098] Some embodiments of the current invention are directed to MEFAs (Microelectrofluidic arrays). Non-limiting examples of embodiments, in addition to those examples and embodiments disclosed herein, can include:

[0099] 1. 3D self-folding shapes with sizes ranging from 0.1 mm to 5 cm with patterns of electrical wires, contact pads and microfluidic channels to simultaneously and independently manipulate and interrogate the chemical and electrical environment in 3D tissue culture and organoids;

[0100] 2 An embodiment where the 3D self-folding shape is a spherical or cubic shell;

[0101] 3. An embodiment where the 3D self-folding shell wraps around live cells and organoids;

[0102] 4. A shell with 5-500 chemical sources (CS) with pore sizes ranging from 10 nm to 1 mm

[0103] 5. A shell capable of creating different chemical patterns including gradients;

[0104] 6. A shell composed of polymers, gel, and metals;

[0105] 7 The multimodal shell capable of electrical, electrochemical, and chemical stimulation and interrogation independently and simultaneously;

[0106] 8. Use of the shell for differentiation of 3D tissue and organoids by delivery of morphogens;

[0107] 9. Use of the shell to test and screen drugs in diseased organoids and 3D tissue; and

[0108] 10. Use of the shell for neuromorphic computation.

[0109] Among the points of novelty associated with systems configured as disclosed herein is the ability to create a tiny (e.g., 10 microns to 1 cm) three-dimensional shape (such as but not limited to a cylinder, a spiral, a polyhedron, or a spherical shell) for which both chemical delivery (e.g., through chemical sources, microfluidic channels, flow controllers, etc.) and electrical stimulation / recording can be achieved. The chemical delivery and electrical stimulation / recording can be done with spatial control (e.g., 1 micron to 1 cm) and temporal control (e.g., timescales from milliseconds to days), using a variety of patterns and gradients with both reactive and unreactive chemicals.

[0110] Devices configured as disclosed herein can include a 3D self-folded device enables wrapping the target shape and conformal contact with it. The 3D channels presented here can be monolithic (embedded within the self-folding photopolymer) and fully fabricated by photolithography. The implementation of origami engineering in these devices enables inverse design of various 3D target shapes that contain microfluidic channels and electrical conduits. Inaddition, a combination of microfluidic channels and electrical conduits in 3D enables electrophoresis,joule heating, electroosmosis, electrochromic reactions, impedance spectroscopy, and other electrically driven phenomena along the channels. Moreover, biocompatibility of the device allows for recording from electrogenic tissues and micro-physiological systems (MPS), stimulating the tissues / MPSs chemically and electrically, monitoring them electrochemically both in situ and ex-situ, ablating a selective part of the tissue chemically or electrically, and extracting chemicals in the tissues from various spatial regions for spatiotemporal monitoring ex-situ and differentiating them with spatiotemporal control.[OHl] Here, we describe self-folding biocompatible shell microfluidic arrays (SMFAs) to enable precise chemical patterning and chemistry within 3D curved microenvironments. We interfaced the SMFAs with programmable flow controllers, enabling precise spatiotemporal delivery of chemicals in a spherical geometry around millimeter-sized 3D structures. We note that this is not just a matter of creating the shell, but also a platform with flow controllers for chemical delivery)

[0112] Moreover, systems configured as disclosed herein provide a reproducible and biocompatible approach for generating dynamic chemical patterns and conducting chemical reactions within 3D microenvironments, with broad relevance to chemistry, embryogenesis, and micro-physiological systems.

[0113] These systems can include customizable spherical microarrays of channels and pores for 3D spatiotemporal chemistry. Additionally, just as chemicals can be dispensed through chemical sources, they can also be retracted by applying negative pressure to the pores within the SMFAs, which could facilitate spatiotemporal micro-sampling of the microenvironments surrounding biological systems and organoids. The biocompatibility of the SMFAs and the ability to tune the 3D spherical spatial localization of Chemical Sources (CSs), combined with the macro-to-microfluidic interface, can enable a variety of morphogen, biochemical, and extracellular signaling molecular patterns.

[0114] FIG. 1 illustrates examples of images of CAD-generated mask designs used in our multilayer photolithographic patterning of the 3D shell microfluidic arrays (SMFAs). UV lithography masks for each layer in the SMFA. The white areas correspond to transparent regions, and the black areas are opaque. SU8 is a negative photoresist; therefore, transparent regions crosslink, while opaque regions remain unexposed and do not crosslink, and are subsequently removedduring development. The illustrated masks have the following purposes: a) Ge sacrificial layer lift-off, b) patterning the first SU8 layer, c) patterning the second SU8 layer, d) patterning the sacrificial filler, e) first exposure (300 mJ / cm2) of the third SU8 layer, and f) second exposure (200 mJ7cm2) of the third SU8 layer. We designed the masks using Autodesk AutoCAD. The designs associated with the example masks can vary according to the specific configuration desired.

[0115] Regarding fabrication of SMFAs, we spin-coated a layer of ~2.7-pm-thick SI 827 photoresist (KAYAKU, Westborough, MA) on a 3-inch silicon wafer (NANOSILICON, USA) at 3000 rpm to create mask patterns for the germanium (Ge) sacrificial layer. We exposed this positive photoresist through a photomask (designed in Autodesk AutoCAD) at an energy density of 150 mJ / cm2to selectively open the windows to the silicon underneath the polymer. We removed the exposed portion of the resist by treating it with MICROPOSIT 351 developer (diluted 1:5 with water, KAYAKU, Westborough, MA) for one minute.

[0116] Then, we deposited a 50-nm-thick Ge layer (Kurt J. Lesker Company, USA) on the silicon wafer through the patterned SI 827 photoresist via thermal evaporation. We employed an acetone lift-off process to define the Ge sacrificial layer, upon which we patterned the selffolding leaflets using a trilayer lithography method (see Fig. 2). We fabricated the first SU8 layer by spin-coating a ~5-pm-thick layer of SU82005 (KAYAKU, Westborough, MA) at 3000 rpm. After a soft baking step (1 min at 65 °C, 3 min at 95 °C, and 1 min at 65 °C, respectively), we selectively crosslinked the negative photoresist layer by exposing it to a total energy density of 240 ml / cm2through a photomask. Subsequently, we post-baked the first SU8 layer (1 min at 65 °C, 3 min at 95 °C, and 1 min at 65 °C, respectively). We developed it in SU8 developer (KAYAKU, Westborough, MA) for 1.5 minutes (see Fig. 2).

[0117] We then spin-coated the second SU8 layer (SU82015, KAYAKU, Westborough, MA), having a thickness of ~16-pm, at 3000 rpm on top of the first SU8 layer and soft-baked the SU8 (1 min, 10 min, and 1 min at 65 °C, 95 °C, and 65 °C, respectively). We patterned the layer by exposing it through a mask at an energy density of 500 mJ / cm2. The exposed layer was baked (1 min, 10 min, and 1 min at 65 °C, 95 °C, and 65 °C, respectively) and then developed in SU8 developer for 2 minutes to obtain the patterned second SU8 layer (see Fig. 2). We then filled the open channels created by the first SU8 layer and the second SU8 layer with a sacrificial filler by spin-coating a layer of positive photoresist SPR220 (KAYAKU, Westborough, MA) at 1700 rpm(see Fig. 2). We exposed this sacrificial filler using the same photomask used for the second SU8 layer (see Fig. 1, parts c and d). Finally, we developed in MICROPOSIT 351 developer (diluted 1:5 with water, KAYAKU, Westborough, MA) for 1 min to create channel filling (see Fig. 2).

[0118] The third SU8 layer in the trilayer lithography process, used to define the roof of the microfluidic channels, was created by spin-coating another layer of SU82015 at 3000 rpm. To make a crosslinking gradient in the trilayer and facilitate self-folding, we exposed it to an energy of 300 mJ / cm2The portion other than the leaflets was fully crosslinked by additionally exposing it to an energy density of 200 mJ / cm2, totaling 500 mJ / cm2. This process forms a solvent-responsive trilayer that folds selectively at the region with a crosslinking gradient (leaflets in the case of SMFA) (see Fig. 2). For the third SU8 layer, we carried out post-exposure baking (1 min at 65 °C, 10 min at 95 °C, and 1 min at 65 °C, respectively) 24 hours after exposure to prevent nitrogen bubble formation from the sacrificial filler. The plate was developed in SU-8 developer for seven days to ensure the complete removal of the sacrificial filler (see Fig. 2). Finally, the Ge sacrificial layer is dissolved in 6% hydrogen peroxide to release the leaflets from the silicon wafer surface (see Fig. 2). Due to the crosslinking gradient of the trilayer at the leaflet region, the trilayer folds when immersed in an aqueous solution following a one-hour acetone treatment (see Fig- 2).

[0119] FIG. 2 illustrates an example schematic of 3D self-folding SMFA fabrication.

[0120] Step 1: Deposit 50 nm of Ge through thermal evaporation and subsequent lift-off in acetone to create a sacrificial layer pattern (mask shown in Fig. 1, item a).

[0121] Step 2: Pattern the first SU8 layer (SU82005) using an appropriate mask (shown in Fig.1, item b) and an exposure energy of 240 mJ / cm2.

[0122] Step 3: Pattern the second SU8 layer (SU82015) using an appropriate mask (shown in Fig. 1, item c) and an exposure energy of 500 mJ / cm2.

[0123] Step 4: Pattern the sacrificial filler (SPR220) using an appropriate mask (shown in Fig. 1, item d) and an exposure energy of 500 mJ / cm2.

[0124] Step 5: Pattern the third SU8 layer (SU82015) using appropriate masks and dual exposure: 300 mJ / cm2for the first exposure (mask shown in Fig. 1, item e) and 200 mJ / cm2for the second exposure (mask shown in Fig. 1, item f).

[0125] Step 6: Remove the sacrificial filler by treating it with SU8 developer.

[0126] Step 7: Dissolve the Ge sacrificial layer in 6% hydrogen peroxide to selectively release the leaflets from the substrate. Self-fold by first treating with acetone, followed by immersing in water. The scale bar is 2 mm.

[0127] FIG. 3 illustrates an example of the removal of a sacrificial filler. Part a illustrates representative images showing removal of the sacrificial filler. We calculated the clearance percentage by estimating the percentage of the total channel area cleared using ImageJ (i.e., the white portion of the channels) from the top-view images, which were converted into a binary image. In part b is illustrated the average clearance of unfolded microfluidic channels of various widths (n=4-5, error bars are one standard deviation), ranging from 50 to 250 pm (inclusive), after 1, 2, and 3 days. All microfluidic channels are 7250 pm long. Wider channels displayed markedly less clearance than narrower channels after one day in developer due to the greater volume of material to be removed. After two days, all channels were over 90% cleared. In part c is illustrated the average clearance of unfolded microfluidic channels of various lengths (n=4-5, error bars are one standard deviation), ranging from 4250 to 10250 pm (inclusive), after 1, 2, and 3 days. All channels are 200 pm wide. Longer channels exhibited significantly less clearance after 24 hours had elapsed. All channels shorter than 8250 pm were cleared after three days; however, the longer channels were still not entirely cleared.

[0128] FIG. 4 illustrates an example of the self-folding of SU8 channels and immersion time in acetone. The top row illustrates an example of self-folding. The middle row illustrates an example of channels exposed to acetone for 1 hr, which caused bending preferentially at the differentially exposed region (roughly halfway down the length of the beam). The bottom row illustrates an example of channels exposed to acetone for 2 hrs, which caused rolling rather than localized bending. The two-hour treatment also caused unwanted delamination of the structure from the silicon substrate (highlighted by the dashed box). Therefore, we used a 1-hour acetone treatment to achieve better control over the fold angle. The scale bar is 1.5 mm.

[0129] FIG. 5 illustrates an example of self-folding behavior with varying width of the differentially exposed fold region (wy). From left to right: We increased the fold region (w / ) by exposing only that portion to 300 mJ / cm2while exposing the rest of the beam to 500 mJ / cm2. Beams with smaller wf, self-fold with smaller fold angles (fold angle is the angle between the beam and the substrate). The scale bar is 3 mm.

[0130] FIG. 6 illustrates an example of 3D self-folding microfluidics with different shapes, with images of uniformly curved and helical self-folded microfluidic devices. The scale bars are 3 mm.

[0131] FIG. 7 illustrates an example fo self-folding synthetic tendrils with different twisting behaviors. We fabricated helical microfluidics that mimic tendrils by creating differentially crosslinked trilayer fold regions at an angle to the long axis of the beam. We achieved different twisting behaviors by manipulating the geometric parameters of the folding region and the total number of folding regions. The scale bar is 3 mm.

[0132] FIG. 8 illustrates an example schematic of the physical bonding methodology used for interfacing external channels with the SMFAs. Stepwise fabrication and integration of SMFAs, microfluidic channels with a PDMS interface.

[0133] Step 1: Mold the PDMS slab.

[0134] Step 2: Punch holes in the PDMS slab using a biopsy punch.

[0135] Step 3: Cut a double-sided adhesive fdm.

[0136] Step 4: Bond one side of the double-sided adhesive to the PDMS slab.

[0137] Step 5: Bond the other side of the double-sided adhesive to the self-folding microfluidic channels.

[0138] Step 6: Insert the microfluidic tubing into the punched holes to interface with external pressure sources.

[0139] FIG. 9 illustrates an example schematic of a chemical bonding methodology used for interfacing external channels with SMFAs. Stepwise fabrication and integration of SMFAs, microfluidic channels with a PDMS interface. In general, the chemical bonding methodology worked better (reduced leakage) as compared to a physical bonding methodology.

[0140] Step 1: Mold a polydimethylsiloxane (PDMS) slab.

[0141] Step 2: Punch holes in the PDMS slab using a biopsy punch.

[0142] Step 3: Treat the part of the SMFA that is in contact with PDMS with (3-aminopropyl) triethoxysilane (APTES).

[0143] Step 4: Bond the PDMS to APTES-treated SU8 after treating the PDMS slab with oxygen plasma and APTES.

[0144] Step 5: Insert the microfluidic tubing into the punched holes to interface with external pressure sources.

[0145] We created the PDMS mold by spin-coating a silicon wafer with SU82025 photoresist (Kayaku, Westborough, MA) at 3000 rpm and patterning it using photolithography with a photomask that featured circular windows where the inlet / outlet should be connected. We then used this as the mold to make PDMS interfacing pieces capable of interfacing with the pump. For molding, we combined Sylgard PDMS base and curing agent (DOW SILICONES CORPORATION; Midland, MI) in a 10: 1 mass ratio and mixed thoroughly. We degassed the mixture in a vacuum chamber and then poured it onto the SU8 mold. The PDMS was placed on a hot plate to cure at 90 °C for 10 minutes, then at 120 °C for 50 minutes. We peeled the molded PDMS off the silicon wafer after the polymer was cured.

[0146] Using a razor blade, we cut the PDMS piece into rectangular slabs, each containing one set of circular depressions laid out by the mask, and punched holes through the PDMS pieces with a biopsy punch. We employed two different bonding strategies to ensure fluidic sealing between the SU8 and the PDMS: a chemical bonding approach using (3 -ami nopropyl) triethoxysilane (APTES) and a physical bonding approach using a double-sided adhesive.Briefly, we started by washing the PDMS piece and SU-8 substrate with isopropyl alcohol (IP A) and then dried with compressed air to ensure the surfaces were clean enough for chemical bonding. We then activated the face of the PDMS piece to be affixed via oxygen bombardment in a plasma etching machine (50 W for 1 minute). Then, the activated PDMS and SU-8 were both immersed in the 5% APTES solution for 20 minutes, after which we cleaned them with DI water, blow-dried, and bonded by heating for 30 minutes at 80°C.

[0147] To create a physical bonding using adhesive, a pattern corresponding to the size of the rectangular PDMS piece and holes corresponding to the position of the circular depressions were etched into a SecureSeal Adhesive Sheet (GRACE BIOLABS; Bend, OR) using a Cricut machine (South Jordan, UT). After removing the plastic-side covering, the adhesive piece was affixed to the PDMS piece, aligning the holes in the adhesive piece with the corresponding holes on the PDMS. The foam side of the adhesive was removed, and the PDMS piece was affixed to its position on the microfluidic plate, aligning the punched holes with the inlets / outlets. After accomplishing either a chemical or physical bonding, microfluidic tubes were pushed into the punched holes and secured using superglue to prevent leakage from the insertion point.

[0148] FIG. 10 illustrates an example of interfacing for perfusion. On the left is an image of the full SMFA setup, depicting interfacing with the PDMS piece and a cloning cylinder for holdingfluid. In the middle is a fluorescent image showing perfusion of SMFA with fluorescent dye. On the right is a close-up image of a single leaflet perfused with dye (green dye in a non-black and white image).

[0149] FIG. 11 illustrates an example of bead placement in an SMFA for spatiotemporal chemical patterning. Images of a three-leaflet SMFA without (left), and with a gel bead (right).

[0150] We used COMSOL computational fluid dynamics (CFD) simulations to verify the feasibility of our constructs in predicting the profile of perfused fluid into a porous medium, such as agarose beads. We constructed a simplified CAD model in SOLIDWORKS, consisting of two concentric spheres: the outer one has three pores representing the shell, and the inner sphere simulates the bead that the shell encapsulates. The model was imported into COMSOL, where we defined the boundary conditions of the CFD simulation.

[0151] We designed our simulation to encompass two primary behaviors: laminar flow and the diffusion of solutes through the liquid surrounding the bead, as well as transport through the bead itself. The outer sphere defines the liquid surrounding the bead. Both laminar flow and diffusion drive the transport of species in this space around the outside of the bead. We simulated this using COMSOL software presets for the fluidic properties of water and diffusion properties of the solute (in our case, Rhodamine 6G) in water. Diffusion through porous media governs the transport of species in this region. To simulate the bead, we defined a porosity coefficient of 0.99 and a diffusion coefficient of 4.1 x IO'10m2 / s in the porous medium COMSOL module We used the Brinkman Equation, considering a permeability value of 616 nm2,3. Additionally, we defined a velocity of u = 0 and a concentration of c = 0 as the initial conditions. The circular openings of each leaflet were enabled or disabled as inlets and outlets, following what we are attempting to simulate. For the case shown in Fig. 21, we used a mass flow rate of 5 pL / min and a solute concentration of 10 mol / m3(for inflows containing the dissolved dye or other molecules). We defined the surface of the outer sphere as an open boundary in the ‘Laminar Flow’ and outflow in the ‘Transport of Diluted Species in Porous Media’ module, through which suspended molecules can diffuse freely out into the surrounding aqueous medium, as in the SFMA, the spaces between the leaflets are open to the surrounding media.

[0152] We conducted a transient simulation to investigate the evolution of the diffusion profile and a static simulation to determine its equilibrium steady state. Fig. 12 shows three cases studied. Case 1 has only one active inlet through which the fluid containing the chemical speciesis delivered, whereas Case 2 has two inlets with the same flow rate, and only one of them has chemical species in it. Case 3 shows the effect of a pulsed flow on the concentration profile of the bead.

[0153] FIG. 12 illustrates an example CFD study illustrating examples of transient and steadystate gradients and pulsed flow. Case 1 has one active inlet and provides a flow rate of 5 pL / min and a concentration of 10 mol / m3. Case 2 has two inlets with the same flow rate (5 uL / min). The inlet on the left has a species concentration of 10 mol / m3, and the one on the right has a concentration of 0 mol / m3. Both transient simulation and static simulation results are shown. Case 1 eventually converges to a uniform concentration throughout the bead, whereas Case 2 results in a sustained gradient. Case 3 illustrates the pattern formation that emerges from a pulsed inlet flow rate.

[0154] FIG. 13 illustrates an example comparison between experiments and simulations of a transient gradient generated in a gel bead using an SMFA. Transient gradient generated inside an agarose bead by delivering a solution through one of the SMFA leaflets. The scale bar is 1 mm.

[0155] FIG. 14 illustrates an example comparison between simulations and experiments of a steady-state gradient in a gel bead generated using an SMFA. We obtained a steady-state gradient by delivering fluid through two inlets and SMFA leaflets. Species 1 is rhodamine 6G, species 2 is methylene blue (non-fluorescent), and species 3 is fluorescein. The scale bar is 1 mm.

[0156] FIG. 15 illustrates examples of a spatiotemporally controlled chemical reaction inside a gel bead using an SMFA. Experimental results showing spatiotemporally controlled magnesium carbonate precipitation inside an agarose bead placed within an SMFA. Part (a) illustrates an experimental image showing precipitation at a single spot, created by diffusing Na2COs through the pore in one leaflet and MgSCU through the pore in an adjacent leaflet. Part (b) illustrates Images of the formation of the product MgCCh pattern as formed towards the interior of the bead, taken at different angles. Part (c) illustrates an experimental image showing precipitation in a different pattern, created by diffusing Na2COs through the pore in one leaflet and MgSC through the pores in both opposing leaflets. Part (d) illustrates images of the formation of a different product MgCCh pattern as formed towards the interior of the bead, taken at various angles. The scale bar is 1 mm.

[0157] FIG. 16 illustrates an example of neural organoid (NO) visibility inside an SFMA. In (a) we illustrate a plot quantifying cytotoxicity using the LDH assay. NOs within the SMFA exhibited a comparable response to those cultured in a Petri dish (control). Row (b) provides examples of Hoechst-stained images of organoids (nuclear dye). The left side represents the bottom, while the right side represents the top of the organoid. Row (c) provides examples of immunostaining for [3-Tubulin Class III, a key marker of early neuronal differentiation, along with the corresponding secondary antibody. The left side represents the bottom, and the right side represents the top of the NO. Row (d) provides examples of immunostaining for Glial Fibrillary Acidic Protein (GFAP), a key astrocyte marker, with the corresponding secondary antibody. The left side represents the bottom, and the right side represents the top of the organoid. Row (e) provides, on the left, an example of a merged image of the organoid, and on the right, a side view of the organoid inside the folded shell. The scale bar is 1 mm.

[0158] FIG. 17 illustrates examples of schematics, fabrication, and operation of the SMFAs. In (a) Conceptual diagram showing spatial chemical patterning of spheres using self-folding SMFAs. Schematics then show the capability of the SMFA to, (b) form spatial gradients, (c) define precise multi-chemical patterns, (d) perform reactions with spatiotemporal control within a gel bead, and (e) selectively stain one organoid within a system of two organoids. In (f) is illustrated a stacked schematic of the different photopattemed layers that form the SMFA. g. A representative experimental image of a multi-colored bead that was patterned within the SMFA. The scale bar indicates 1 mm.

[0159] FIG. 18 illustrates an example of wafer scale fabrication and self-folding characteristics of SMFAs. In (a) is provided a photograph of 48 single-channel microfluidic patterns on a three-inch wafer indicating parallel, batch fabrication, (b) illustrates a zoomed-in image of the channels on the wafer after self-folding with specific regions curved while others lying flat to interface with external controls. Scale bar is 5 mm. In (c) is provided a plot of fold angle vs. fold width. In our study, the extent of folding is tuned by tuning the fold width. In (d) schematics and experimentally realized images are illustrated showing that multiple bending regions can be arranged to manipulate the overall folding characteristics. Also, multiple fold regions can be patterned at varying angles with the long axis to cause self-folding with uniform and helical radii of curvature. Also, smaller ratios of fold-to-strip width (wfAv) result in a larger overall helical pitch.

[0160] FIG. 19 illustrates an example of self-folding microfluidics with bioinspired shapes. In (a) is illustrated a photograph of a self-folded helical microfluidic channel. Multiple selective exposures over a tilted area form helical microfluidic structures. In (b) is illustrated an image of a self-folded cylindrical microfluidic structure containing multiple microchannels. In (c) and (d), are illustrated photographs of SMFAs with different curvature and three and five channels respectively. All scale bars represent 1 mm.

[0161] FIG. 20 illustrates an example of controlled 3D multi-chemical microfluidic dispensation through SMFAs. Chemical perfusion through self-folded microfluidics with a) one, b) three, and c) five 3D microchannels. Scale bars are 1 mm. d. Controlled sequential chemical perfusion (yellow, red, and green in order) through each leaflet of the three leaflet SMFA. The scale bars are 5 mm. Insets show zoomed-in images of the leaflets. The scale bars in the zoomed-in images are 1 mm.

[0162] FIG. 21 illustrates an example of CFD-assisted gradient pattering of gel beads using SMFAs. At the top, in (a), is illustrated a comparison between CFD simulations and experimental results at various time points (0 s, 120 s, 600 s, 1200 s) showing the transient chemical gradient within the bead generated by selective perfusion through one pore of an SMFA. The scale bars are 1 mm. At the bottom, in (b), are illustrated simulations and experiments showing how introducing two different chemical solutions through different pores creates a steady state gradient. The scale bar is 1 mm.

[0163] FIG. 22 illustrates an example of multi-chemical localized spatial gradient of gel beads. Sequential controlled chemical perfusion of blue, red, and green solutions is shown over time (1 min, 7 min, and 25 min). The scale bars are 750 pm.

[0164] FIG. 23 illustrates examples of 3D microchemistry inside a gel bead by spatiotemporal release of reactants using SMFAs. Schematic, simulation and experimental results demonstrating the localized reaction between NaaCCh and MgSC>4 (reactants) to form MgCOs precipitates (product) inside a gel bead. In (a) is illustrated a diffusion front from a chemical source (CS1) of Na2CC>3, reacting with the one of MgSCh (CS2) to precipitates MgCOs (white) inside the gel bead. Concentration profile of each reactant and the iso-surface of the product of the concentration are shown in the first row. The corresponding experimental demonstration using SMFA is shown in the second row. In (b) are illustrated diffusion fronts from two chemical sources (CS2 and CS3) of MgSC>4, reacting with source Na2COs (CS1), resulting in two distinctMgCCh dots, which are later merged. Concentration profde of each reactant and the iso-surface of the product of the concentration are shown in the first row. The corresponding experimental demonstration using SMFA is shown in the second row. Dotted lines outline the boundaries of the gel bead. The scale bar is 1 mm.

[0165] FIG. 24 illustrates an example of a biocompatibility assessment and selective straining of neural organoids (NOs). In (a) a plot shows the cell viability assessed using resazurin. Results show that cell viability in the presence of the SMFA is comparable to controls cultured in Petri dishes. In (b) we see fluorescent images of neurite bridges formed between the organoid (yellow line) and the SMFA wall (white line) after a week in the devices. In (c) at the top a schematic shows selectively staining of one NO within a two-organoid system placed within the SMFA. In (c) at the bottom are shown experimental results showing a two-NO system where the Hoechst 33342 trihydrochloride (in blue) is selectively supplied through leaflet 1 of the SMFA only towards NO1 while media is supplied through leaflet 2 of the SMFA towards NO2 resulting in selective staining of NO1. The scale bar is 750 pm.

[0166] FIG. 25 illustrates an example of a Multimodal microelectronic fluidic array (MEFA) fabrication process flow. This process is similar to the process illustrated in FIG. 2. Here, the process illustrated shows the steps involved in the fabrication of multimodal MEFA and the corresponding microscope images.

[0167] FIG. 26 illustrates examples of potential applications of MEFAs. Non-limiting examples of MEFAs can include electrical modulation (e.g., 3D electrochromic reactions and monitoring), chemical modulation (e.g., 3D chemical infusion and chemical monitoring), and / or biological modulation (e.g., 3D chemical feedback and electrophysiology).

[0168] FIG. 27 illustrates an example of a multimodal MEFA. As illustrated, the multimodal MEFA shell uses a combination of electrical and chemical stimulation on organoids. The combination enables the study of spontaneous neurochemical secretion and the ones resulting from electrical and chemical stimulation. The combination also enables spatiotemporally controlled delivery of chemicals such as morphogens and how that affect the chemical and electrophysiological behavior of the organoid at different locations at various time points.

[0169] FIG. 28 illustrates examples of fluidic and electrical interfacing with a multimodal MEFA. As illustrated, the feedback recorded by analyzing the electrophysiology can be used to provide positive or negative chemical reinforcement in a circular process.

[0170] With reference to FIG. 29, an exemplary system includes a computing device 2900 (such as a general-purpose computing device), including a processing unit (CPU or processor) 2920 and a system bus 2910 that couples various system components including the system memory 2930 such as read-only memory (ROM) 2940 and random access memory (RAM) 2950 to the processor 2920. The computing device 2900 can include a cache of high-speed memory connected directly with, in close proximity to, or integrated as part of the processor 2920. The computing device 2900 copies data from the system memory 2930 and / or the storage device 2960 to the cache for quick access by the processor 2920. In this way, the cache provides a performance boost that avoids processor 2920 delays while waiting for data. These and other modules can control or be configured to control the processor 2920 to perform various actions. Other system memory 2930 may be available for use as well. The system memory 2930 can include multiple different types of memory with different performance characteristics. It can be appreciated that the disclosure may operate on a computing device 2900 with more than one processor 2920 or on a group or cluster of computing devices networked together to provide greater processing capability. The processor 2920 can include any general-purpose processor and a hardware module or software module, such as module 1 2962, module 22964, and module 3 2966 stored in storage device 2960, configured to control the processor 2920 as well as a special-purpose processor where software instructions are incorporated into the actual processor design. The processor 2920 may essentially be a completely self-contained computing system, containing multiple cores or processors, a bus, memory controller, cache, etc. A multi-core processor may be symmetric or asymmetric.

[0171] The system bus 2910 may be any of several types of bus structures including a memory bus or memory controller, a peripheral bus, and a local bus using any of a variety of bus architectures. A basic input / output (BIOS) stored in memory ROM 2940 or the like, may provide the basic routine that helps to transfer information between elements within the computing device 2900, such as during start-up. The computing device 2900 further includes storage devices 2960 such as a hard disk drive, a magnetic disk drive, an optical disk drive, tape drive or the like. The storage device 2960 can include software modules 2962, 2964, 2966 for controlling the processor 2920. Other hardware or software modules are contemplated. The storage device 2960 is connected to the system bus 2910 by a drive interface. The drives and the associated computer-readable storage media provide nonvolatile storage of computer-readableinstructions, data structures, program modules and other data for the computing device 2900. In one aspect, a hardware module that performs a particular function includes the software component stored in a tangible computer-readable storage medium in connection with the necessary hardware components, such as the processor 2920, system bus 2910, output device 2970 (such as a display or speaker), and so forth, to carry out the function. In another aspect, the system can use a processor and computer-readable storage medium to store instructions which, when executed by a processor (e.g., one or more processors), cause the processor to perform a method or other specific actions. The basic components and appropriate variations are contemplated depending on the type of device, such as whether the computing device 2900 is a small, handheld computing device, a desktop computer, or a computer server.

[0172] Although the exemplary embodiment described herein employs the storage device 2960 (such as a hard disk), other types of computer-readable media which can store data that are accessible by a computer, such as magnetic cassettes, flash memory cards, digital versatile disks, cartridges, random access memories (RAMs) 2950, and read-only memory (ROM) 2940, may also be used in the exemplary operating environment. Tangible computer-readable storage media, computer-readable storage devices, or computer-readable memory devices, expressly exclude media such as transitory waves, energy, carrier signals, electromagnetic waves, and signals per se.

[0173] To enable user interaction with the computing device 2900, an input device 2990 represents any number of input mechanisms, such as a microphone for speech, a touch-sensitive screen for gesture or graphical input, keyboard, mouse, motion input, speech and so forth. An output device 2970 can also be one or more of a number of output mechanisms known to those of skill in the art. In some instances, multimodal systems enable a user to provide multiple types of input to communicate with the computing device 2900. The communications interface 2980 generally governs and manages the user input and system output. There is no restriction on operating on any particular hardware arrangement and therefore the basic features here may easily be substituted for improved hardware or firmware arrangements as they are developed.

[0174] The computing device 2900 may be described in the general context of computer systemexecutable instructions, such as program modules, being executed by a computer system.Generally, program modules may include routines, programs, objects, components, logic, data structures, and so on that perform particular tasks or implement particular abstract data types. Inconfigurations where the computing device 2900 is used in a distributed cloud computing environment (such as where the computing device 2900 utilizes one or more servers) where tasks are performed by remote processing devices that are linked through a communications network. In a distributed cloud computing environment, program modules may be located in both local and remote computer system storage media including memory storage devices.

[0175] The technology discussed herein refers to computer-based systems and actions taken by, and information sent to and from, computer-based systems. One of ordinary skill in the art will recognize that the inherent flexibility of computer-based systems allows for a great variety of possible configurations, combinations, and divisions of tasks and functionality between and among components. For instance, processes discussed herein can be implemented using a single computing device or multiple computing devices working in combination. Databases, memory, instructions, and applications can be implemented on a single system or distributed across multiple systems. Distributed components can operate sequentially or in parallel.

[0176] Use of language such as “at least one of X, Y, and Z,” “at least one of X, Y, or Z,” “at least one or more of X, Y, and Z,” “at least one or more of X, Y, or Z,” “at least one or more of X, Y, and / or Z,” or “at least one of X, Y, and / or Z,” are intended to be inclusive of both a single item (e.g., just X, or just Y, or just Z) and multiple items (e.g., {X and Y), {X and Z), {Y and Z}, or {X, Y, and Z }). The phrase “at least one of’ and similar phrases are not intended to convey a requirement that each possible item must be present, although each possible item may be present. The term “approximately” can mean close to a particular time, measurement, or number — but it's not exact. As a non-limiting example, approximately can mean within + / - 10% of a given value.

[0177] The various embodiments described above are provided by way of illustration only and should not be construed to limit the scope of the disclosure. Various modifications and changes may be made to the principles described herein without following the example embodiments and applications illustrated and described herein, and without departing from the spirit and scope of the disclosure. For example, unless otherwise explicitly indicated, the steps of a process or method may be performed in an order other than the example embodiments discussed above. Likewise, unless otherwise indicated, various components may be omitted, substituted, or arranged in a configuration other than the example embodiments discussed above.

[0178] Further aspects of the present disclosure are provided by the subject matter of the following clauses.

[0179] A method, comprising: creating, using photolithography, an initial wafer containing: at least one gradient crosslinked self-folding film layer; and at least one sacrificial filler layer; and dissolving the at least one sacrificial filler layer of the initial wafer, resulting in a self-folding biocompatible shell microfluidic array (SMFA) comprising at least one microfl ui die channel.

[0180] The method of any preceding clause, the creating of the initial wafer further comprises: applying a first photomask to a wafer which defines a thermally evaporated germanium sacrificial layer; applying a second photomask, a third photomask, and a fourth photomask of photoresist film to the wafer to define layers of the at least one gradient crosslinked self-folding film layer, thereby forming a trilayer, and further defining a shape of the at least one microfluidic channel; applying a fifth photomask to the wafer which defines the at least one sacrificial filler layer; applying a sixth photomask to the wafer which adds an additional layer of the photoresist film to a first portion of the trilayer; after applying the sixth photomask, applying an Ultra Violet (UV) light to the wafer such that the wafer comprises a folding region distinct from the first portion, the folding region having less exposure to the UV light than the first portion, resulting in the initial wafer.

[0181] The method of any preceding clause, further comprising: immersing the SMFA in acetone, resulting in self-folding of the SMFA.

[0182] The method of any preceding clause, wherein immersion of the SMFA in acetone occurs for approximately 1 minute.

[0183] The method of any preceding clause, wherein the SMFA has a size within a range of 0.1 mm to 5 cm, with patterns of electrical wires, contact pads and the at least one microfluidic channel to simultaneously and independently manipulate and interrogate a chemical and electrical environment in 3D tissue culture and organoids.

[0184] The method of any preceding clause, wherein the dissolving of the at least one sacrificial filler layer comprises placing the initial wafer in a solution comprising hydrogen peroxide.

[0185] The method of any preceding clause, wherein the dissolving of the at least one sacrificial filler layer releases leaflets to self-fold.

[0186] The method of any preceding clause, wherein the leaflets comprise a trilayer of the at least one gradient crosslinked self-folding film layer.

[0187] The method of any preceding clause, wherein the at least one sacrificial filler layer is defined using a lift-off process.

[0188] The method of any preceding clause, further comprising: inputting chemical sources into the SMFA via the at least one microfluidic channel.

[0189] The method of any preceding clause, wherein a gradient of the at least one gradient crosslinked self-folding film layer comprises one of: a steady-state gradient; a transient gradient; or a spatial gradient.

[0190] The method of any preceding clause, wherein the gradient forms upon exposing the initial wafer to Ultra Violet (UV) energy.

[0191] The method of cany preceding clause, wherein the SMFA is configured for simultaneous fluidic stimulation, electrical stimulation, and / or recording.

[0192] A self-folding biocompatible shell microfluidic array (SMFA) comprising: gradient crosslinked self-folding film layers with embedded fillers; and at least one hollow microfluidic channel, wherein the at least one hollow microfluidic channel have widths as small as 200 pm.

[0193] The SMFA of any preceding clause, wherein: the gradient crosslinked self-folding film layers self-fold to form at least one of a cylinder, a spiral, or a spherical shell.

[0194] The SMFA of any preceding clause, wherein the at least one hollow microfluidic channel comprises at least one inlet and at least one outlet.

[0195] The SMFA of any preceding clause, wherein the at least one inlet and the at least one outlet are bonded to an external fluidic instrumentation using plasma bonding process assisted by (3 -aminopropyl) triethoxysilane (APTES).

[0196] The SMFA of any preceding clause, wherein a gradient of the gradient crosslinked selffolding fdm layers comprises one of: a steady-state gradient; a transient gradient; or a spatial gradient.

[0197] The SMFA of any preceding clause, wherein the gradient forms upon exposure of a selffolding fdm layer to Ultra Violet (UV) energy.

[0198] The SMFA of any preceding clause, wherein the SMFA is configured for simultaneous fluidic stimulation, electrical stimulation, and / or recording.

[0199] A multimodal self-folding biocompatible shell microfluidic array (SMFA) comprising: gradient crosslinked self-folding film layers with embedded fillers; at least one hollow microfluidic channel; electrical wiring: and contact pads coupled to the electrical wiring.

[0200] The multimodal SMFA of any preceding clause, wherein the multimodal SMFA is configured for simultaneous fluidic stimulation, electrical stimulation, and / or recording.

[0201] A self-folding biocompatible shell microfluidic array (SMFA) comprising: gradient or multilayer differentially crosslinked self-folding film layers; at least one leaflet; and at least one hollow microfluidic channel, wherein the at least one hollow microfluidic channel provides connection to at least one chemical source or sink connected to the at least one hollow microfluidic channel.

[0202] The SMFA of any preceding clause, further comprising: a digital microfluidic flow controller; a pressure sensor; and a flow sensor.

[0203] The SMFA of any preceding clause, wherein: the gradient or multilayer differentially crosslinked self-folding film layers self-fold to form a three-dimensional (3D) shape, the 3D shape comprising at least one of a cylinder, a spiral, a polyhedron, or a spherical shell.

[0204] The SMFA of any preceding clause, wherein: the SMFA is configured for both chemical delivery and chemical removal in a desired pattern in three-dimensions around an object.

[0205] The SMFA of any preceding clause, wherein the SMFA is configured for delivery and generation of chemical patterns and gradients of both reactive chemicals and unreactive chemicals within the SMFA.

[0206] The SMFA of any preceding clause, wherein the generation of the chemical patterns and gradients occurs with both spatial and temporal control.

[0207] The SMFA of any preceding clause, wherein the at least one hollow microfluidic channel comprises at least one inlet and at least one outlet.

[0208] The SMFA of any preceding clause, wherein the at least one inlet and the at least one outlet are bonded to an external fluidic instrumentation using plasma bonding process assisted by (3 -aminopropyl) triethoxysilane (APTES).

[0209] The SMFA of any preceding clause, wherein the SMFA forms a sphere, the SMFA further comprising: a plurality of chemical sources arranged around the sphere, wherein the plurality of chemical sources can be released into the SMFA with spatiotemporal control in three dimensions.

[0210] The SMFA of any preceding clause, wherein release of the plurality of chemical sources results in a chemical gradient within the SMFA, the chemical gradient comprising one of: a steady-state gradient; a transient gradient; or a spatial gradient.

[0211] The SMFA of any preceding clause, the SMFA encapsulating at least one of live cells or organoids.

[0212] The SMFA of any preceding clause, wherein the SMFA is configured to perform at least one of spatiotemporal interrogation and differentiation.

[0213] A multimodal self-folding biocompatible shell microfluidic array (SMFA) comprising: gradient crosslinked self-folding film layers with embedded fillers; at least one hollow microfluidic channel; electrical wiring: and contact pads coupled to the electrical wiring.

[0214] The multimodal SMFA of any preceding clause, wherein the multimodal SMFA is configured for simultaneous fluidic stimulation, electrical, electrophsyiological stimulation, electrochemical stimulation, and recording.

[0215] An automated shell microfluidic array (SMFA) multimodal shell, comprising: gradient or multilayer differentially crosslinked self-folding film layers; at least one leaflet; and at least one hollow microfluidic channel, wherein the at least one hollow microfluidic channel provides connection to at least one chemical source or sink connected to the at least one hollow microfluidic channel; and wherein the at least one microfluidic channel are configured to provide feedback between (1) a macrointerface and (2) controllers connected to chemical sources and electrodes. Non-limiting examples of macrointerfaces can include computer systems configured to interact with the SMFA through the controllers using computer programs, algorithms, neural networks, etc.

[0216] The automated SMFA multimodal shell of any preceding clause, further comprising: a digital microfluidic flow controller; a pressure sensor; and a flow sensor.

[0217] The automated SMFA multimodal shell of any preceding clause, wherein: the gradient or multilayer differentially crosslinked self-folding fdm layers self-fold to form a three-dimensional (3D) shape, the 3D shape comprising at least one of a cylinder, a spiral, a polyhedron, or a spherical shell.

[0218] The automated SMFA multimodal shell of any preceding clause, wherein: the SMFA is configured for both chemical delivery and chemical removal in a desired pattern in three-dimensions around an object.

[0219] The automated SMFA multimodal shell of any preceding clause, wherein the SMFA is configured for delivery and generation of chemical patterns and gradients of both reactive chemicals and unreactive chemicals within the shell.

[0220] The automated SMFA multimodal shell of any preceding clause, wherein the at least one hollow microfluidic channel comprises at least one inlet and at least one outlet.

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Claims

CLAIMSWe claim:

1. A self-folding biocompatible shell microfluidic array (SMFA) comprising:gradient or multilayer differentially crosslinked self-folding film layers;at least one leaflet; andat least one hollow microfluidic channel,wherein the at least one hollow microfluidic channel provides connection to at least one chemical source or sink connected to the at least one hollow microfluidic channel.

2. The SMFA of claim 1, further comprising:a digital microfluidic flow controller;a pressure sensor; anda flow sensor.

3. The SMFA of claim 1, wherein:the gradient or multilayer differentially crosslinked self-folding film layers self-fold to form a three-dimensional (3D) shape, the 3D shape comprising at least one of a cylinder, a spiral, a polyhedron, or a spherical shell.

4. The SMFA of claim 3, wherein:the SMFA is configured for both chemical delivery and chemical removal in a desired pattern in three-dimensions around an object.

5. The SMFA of claim 4, wherein the SMFA is configured for delivery and generation of chemical patterns and gradients of both reactive chemicals and unreactive chemicals within the SMFA.

6. The SMFA of claim 5, wherein the generation of the chemical patterns and gradients occurs with both spatial and temporal control.

7. The SMFA of claim 1, wherein the at least one hollow microfluidic channel comprises at least one inlet and at least one outlet.

8. The SMFA of claim 7, wherein the at least one inlet and the at least one outlet are bonded to an external fluidic instrumentation using plasma bonding process assisted by (3 -aminopropyl) triethoxysilane (APTES).

9. The SMFA of claim 1, wherein the SMFA forms a sphere, the SMFA further comprising:a plurality of chemical sources arranged around the sphere,wherein the plurality of chemical sources can be released into the SMFA with spatiotemporal control in three dimensions.

10. The SMFA of claim 9, wherein release of the plurality of chemical sources results in a chemical gradient within the SMFA, the chemical gradient comprising one of:a steady- state gradient;a transient gradient; ora spatial gradient.11 The SMFA of claim 1, the SMFA encapsulating at least one of live cells or organoids.

12. The SMFA of claim 11, wherein the SMFA is configured to perform at least one of spatiotemporal interrogation and differentiation.

13. A multimodal self-folding biocompatible shell microfluidic array (SMFA) comprising:gradient crosslinked self-folding film layers with embedded fillers;at least one hollow microfluidic channel;electrical wiring: andcontact pads coupled to the electrical wiring.

14. The multimodal SMFA of claim 13, wherein the multimodal SMFA is configured for simultaneous fluidic stimulation, electrical, electrophsyiological stimulation, electrochemical stimulation, and recording.

15. An automated shell microfluidic array (SMFA) multimodal shell, comprising:gradient or multilayer differentially crosslinked self-folding film layers;at least one leaflet; andat least one hollow microfluidic channel,wherein the at least one hollow microfluidic channel provides connection to at least one chemical source or sink connected to the at least one hollow microfluidic channel; and wherein the at least one microfluidic channel are configured to provide feedback between (1) a macrointerface and (2) controllers connected to chemical sources and electrodes.

16. The automated SMFA multimodal shell of claim 15, further comprising:a digital microfluidic flow controller;a pressure sensor; anda flow sensor.

17. The automated SMFA multimodal shell of claim 15, wherein:the gradient or multilayer differentially crosslinked self-folding film layers self-fold to form a three-dimensional (3D) shape, the 3D shape comprising at least one of a cylinder, a spiral, a polyhedron, or a spherical shell.

18. The automated SMFA multimodal shell of claim 17, wherein:the SMFA is configured for both chemical delivery and chemical removal in a desired pattern in three-dimensions around an object.

19. The automated SMFA multimodal shell of claim 18, wherein the SMFA is configured for delivery and generation of chemical patterns and gradients of both reactive chemicals and unreactive chemicals within the shell.

20. The automated SMFA multimodal shell of claim 15, wherein the at least one hollow microfluidic channel comprises at least one inlet and at least one outlet.

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