WO2025199189A1

WO2025199189A1 - Systems and methods of implantation of bioelectronics

Info

Publication number: WO2025199189A1
Application number: PCT/US2025/020504
Authority: WO
Inventors: Jia Liu; Hao SHENG; Ariel LEE
Original assignee: Harvard University
Current assignee: Harvard University
Priority date: 2024-03-20
Filing date: 2025-03-19
Publication date: 2025-09-25
Anticipated expiration: 2026-09-20

Abstract

The present disclosure generally relates to nanoscale wires and nanoelectronics. Certain non-limiting embodiments are generally directed to implantation of stretchable electronics, e.g., in organisms, such as juveniles. For example, one aspect is generally directed to a stretchable component, which may form a part of an electrical circuit. The component may be connectable in certain embodiments to an external device, e.g., to determine a property of the component (e.g., an electrical property), and/or to apply a stimulus (e.g., an electrical stimulus) to a tissue in the organism. Other aspects are generally directed to methods of making or using such components, kits including such components, biological structures containing such components, or the like.

Description

SYSTEMS AND METHODS OF IMPLANTATION OF BIOELECTRONICS

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/567,834, filed March 20, 2024, entitled “Systems and Methods of Implantation of Bioelectronics,” by Liu, et al. ; and of U.S. Provisional Patent Application Serial No. 63/681,224, filed August 9, 2024, entitled “Systems and Methods of Implantation of Bioelectronics,” by Liu, et al. ; each of which is incorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under MH123948 awarded by National Institutes of Health (NIH). The government has certain rights in this invention.

FIELD

The present disclosure generally relates to nanoscale wires and nanoelectronics. Certain embodiments are generally directed to implantation of stretchable electronics, e.g., in organisms, such as juveniles.

BACKGROUND

Recording of neural activities throughout the course of brain development is important to understanding how neurons self-assemble into an organ capable of learning, behavior, and cognition. To date, however, it has not been possible to conduct the ideal experiment: recording brain-wide neural activity at the cellular level with millisecond temporal resolution in animals throughout the course of brain development. For example, functional magnetic resonance imaging (fMRI) acquires brain-wide activity non-invasively but has a low spatiotemporal resolution, and long-term imaging is challenging. Microscopic imaging of genetically encoded calcium or voltage reporters can allow tissue-wide, cellspecific imaging. Using such tools, the brain-wide calcium activity of a few transparent species (e.g., larval zebrafish) has been studied. Tissue scattering and 3D volumetric scanning, however, limit fast optical access to cells throughout 3D brains in non-transparent animals.

Implanted microelectrodes have the potential to overcome these limitations. Existing technologies support the interrogation of single-unit activity with millisecond temporal resolution in 3D tissue. Multielectrode arrays can simultaneously track electrical activities from a large number of neurons in the brain without sacrificing spatial or temporal resolution. The developing brain, however, presents additional challenges. Vertebrate brains are complex 3D structures which originate from a 2D single-cell layer in the embryo. During development, cell division, proliferation, and folding events result in rapid volume expansion and large changes in tissue morphology. Conventional microelectronics cannot interrogate such dynamically changing environments with accuracy, compatibility, and longevity because of fundamental mechanical mismatches between electronic and biological materials.

Recent advances in flexible electronics have yielded “tissue-like” mesh microelectronics: sub-micrometer-thick mesh electronics with tissue-level flexibility. Mesh microelectronics mimics the physicochemical properties of the extracellular matrix and are thus capable of seamlessly integrating into in vitro and in vivo neural tissues, forming gliosis- free bioelectronic interfaces and enabling long-term, stable, multichannel electrical recordings in 3D brain tissues at single-unit, single- spike spatiotemporal resolution over months. Neurons, however, are innervated at the nanometer scale. Regardless of how small and soft bioelectronics are designed to be, the implantation of such devices into the mature brain necessarily introduces acute damage to neural tissue. Accordingly, improvements are needed.

SUMMARY

The present disclosure generally relates to nanoscale wires and nanoelectronics. In some aspects, these may be embedded in biological structures, such as tissues, organoids, organs, organisms, and the like. Certain embodiments are generally directed to implantation of stretchable electronics, e.g., in organisms, such as juveniles. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

The design of bioelectronics capable of stably tracking brain- wide, single-cell, and millisecond-resolved neural activities in the developing brain is important to the study of neuroscience and neurodevelopmental disorders. During development, the three-dimensional (3D) structure of the vertebrate brain arises from a 2D neural plate. These large morphological changes previously posed a challenge for implantable bioelectronics to track neural activity throughout brain development. Certain embodiments are directed to a tissue- level-soft, sub-micrometer-thick, stretchable mesh microelectrode array capable of integrating into the embryonic neural plate (e.g., frog, axolotl, mouse, etc.) by leveraging the 2D-to-3D reconfiguration process of the tissue itself. Driven by the expansion and folding processes of organogenesis, the stretchable mesh electrode array may deform, stretch, and/or distribute throughout the brain, which may fully integrate into the 3D tissue structure. Immunostaining, gene expression analysis, and behavioral testing show no discernable impact on brain development or function. The embedded electrode array may allow, for example, long-term, stable, brain-wide, single-unit- single- spike-resolved electrical mapping throughout brain development, illustrating how neural electrical activities and population dynamics emerge and evolve during brain development. In some embodiments, stretchable electronics can be integrated into developing biological tissue in vitro via normal developmental processes. The forces exerted by the developing tissue may unfold and reconfigure the bioelectronics, distributing sensors throughout the 3D structure.

Some embodiments are directed to various bioelectronic devices and methods to integrate the device within the developing brain of vertebrate animals throughout embryogenesis. Certain embodiments are directed to sub-micrometer- thick, tissue-level soft mesh electronics containing a stretchable electrode array that can be implanted into the embryo neural plate. During organogenesis, the neural plate undergoes a 2D-to-3D reorganization process, folding, proliferating, and expanding into the precursors of the nervous system. The endogenous forces involved in this process may, in certain cases, seamlessly and non-invasively distribute and integrate the sensor network across the 3D volume of the neural tube and brain, creating a “cyborg” embryo. In some cases, the presence of microelectronics may have no discernible impact on embryo development or subsequent behaviors. Certain embodiments are directed to tissue-wide, continuous recording of neuron electrical activity at millisecond temporal resolution. For example, in some embodiments, a device may be used to illustrate the emergence of electrical activities in the neural plate and the evolution, synchronization, and propagation of neural dynamics over the time course of embryonic brain development.

One set of embodiments is generally directed to a method comprising implanting a stretchable electronic component in a tissue in a juvenile organism, the electronic component defining at least a portion of an electrical circuit, and determining an electronic signal from the electrical circuit over a time interval, for example, of at least 2 weeks.

Another set of embodiments is generally directed to a method, comprising implanting a stretchable electronic component in a tissue in an organism using a guide, and removing the guide from the tissue, e.g., without removing the electronic component.

Yet another set of embodiments is generally directed to an article comprising a nonhuman organism having a brain, the brain comprising a stretchable cell scaffold therein, wherein the cell scaffold defines at least a portion of an electrical circuit. Another set of embodiments is generally directed to a method comprising inserting, into a neural plate of an embryo, a stretchable cell scaffold, wherein the cell scaffold defines at least a portion of an electrical circuit.

Another set of embodiments is generally directed to a method comprising exposing a neural plate of an embryo to a stretchable cell scaffold defining at least a portion of an electrical circuit; and growing the embryo to form an organism containing an embedded cell scaffold therein in its brain.

Another set of embodiments is generally directed to a method comprising applying an electrical signal to at least a portion of a brain of an organism using a stretchable cell scaffold embedded within the brain of the organism.

Another set of embodiments is generally directed to a method comprising determining an electrical property of at least a portion of a brain of an organism at a resolution of at least 1 mm using a stretchable cell scaffold embedded within the brain of the organism.

In another aspect, the present disclosure encompasses methods of making one or more of the embodiments described herein, for example, a biological structure embedding a flexible cell scaffold containing electronics, such as a nanoelectronic mesh. In still another aspect, the present disclosure encompasses methods of using one or more of the embodiments described herein, for example, a biological structure embedding a flexible cell scaffold containing electronics, such as a nanoelectronic mesh.

In addition, certain non-limiting embodiments are generally directed to brain implantation of tissue-level-soft and stretchable bioelectronics via embryonic development. For example, one aspect is generally directed to stretchable cell scaffolds, which may be connected to electrical circuits. In some cases, the scaffold may be implanted into a neural plate of an embryo, which may develop to form the brain of the growing organism, e.g., thereby causing the cell scaffold to become embedded within the brain of the organism. The scaffold may be connectable in certain embodiments to an external device, e.g., to determine a property of the cell scaffold (e.g., an electrical property), and/or to apply a stimulus (e.g., an electrical stimulus) to the biological structure. Other aspects are generally directed to methods of making or using such cell scaffolds, kits including such cell scaffolds, biological structures containing such cell scaffolds, or the like.

Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

FIG. 1 illustrates a device that comprises components such as a plurality of nodes connected by various interconnects, e.g., forming a mesh structure, using an embodiment;

FIG. 2 illustrates interconnects that may have an “S” or a serpentine shape, according to some embodiments;

FIG. 3 illustrates a chromium/gold/chromium deposition process in accordance with certain embodiments;

FIGs. 4A-4K illustrate a design of soft and stretchable bioelectronics for brain implantation via embryonic development, in one embodiment;

FIGs. 5A-5M illustrate fabrication of tissue-level-soft stretchable mesh microelectronics for brain implantation via embryo development, in another embodiment;

FIGs. 6A-6D illustrate fabrication of tissue-level-soft stretchable mesh microelectronics for brain implantation via embryo development, in another embodiment;

FIGs. 7A-7Q illustrate a minimally invasive brain implantation of tissue-level-soft, stretchable mesh microelectronics via embryonic development, in yet another embodiment;

FIGs. 8A-8O illustrate continuous tracking of in vivo neural electrical activities from the same tadpole during organogenesis and brain development, in still another embodiment;

FIGs. 9A-9P illustrate implantation and continuous recording of axolotl embryos with soft and stretchable bioelectronics with high-density mesh electrodes, in yet another embodiment, in another embodiment;

FIGs. 10A-10H show the investigation of neural processing of distant injury and tail regeneration using high density electrode arrays in late-stage axolotl embryos;

FIGs. 11A-1 IL illustrate a test of various implantation methods, in certain embodiments;

FIGs. 12A-12B illustrate mechanical simulation of stretchable mesh for brain implantation via embryo development, in one embodiment; FIGs. 13A-13H illustrate fabrication of PFPE-DMA encapsulated stretchable mesh microelectronics, in another embodiment;

FIGs. 14A-14O illustrate a characterization of PFPE-DMA encapsulated stretchable mesh microelectronics, in still another embodiment;

FIGs. 15A-15E illustrate staining methods and extended immunofluorescence images, in accordance with certain embodiments;

FIGs. 16A-16L illustrate experimental setup, trajectory analysis, and examples of behavior tests, in accordance with various embodiments;

FIGs. 17A-17I illustrate an experimental setup, raw data, and reference comparison of continuous electrophysiology, in certain embodiments;

FIGs. 18A-18T illustrate extended data and analysis of continuous electrophysiology, in certain embodiments;

FIGs. 19A-19I illustrate high density mesh electrode array enabling traces of single units, in one set of embodiments;

FIGs. 20A-20C illustrate ISI, autocorrelation and waveform dynamics in axolotl recordings, in various embodiments;

FIGs. 21A-21E show single-unit action potential in axolotl embryonic brain development, in some embodiments;

FIGs. 22A and 22B illustrate structures of PFPE-DMA embryo device in certain embodiments;

FIGs. 23A-23C illustrate long term rearing of cyborg tadpoles to cyborg frogs, in some embodiments;

FIGs. 24A and 24B illustrate immuno staining images depicting the contact between tissues and mesh electronics, in one set of embodiments;

FIGs. 25A and 25B illustrate a quantitative analysis procedures of fluorescence images, in some embodiments;

FIG. 26 illustrates a design of a stretchable device with a 32-channel tetrode-like mesh electrode array, in another embodiment;

FIGs. 27A-27F illustrate a neurulation implantation of PFPE-DMA mesh microelectronics in mouse embryos, in yet another embodiment;

FIGs. 28A-28C illustrate agarose fixation of cyborg frog tadpole, in still another embodiment;

FIGs. 29A-29F illustrate an electrophysiological recording of cyborg frog tadpole with 16-channel electrode array and agarose fixation, in yet another embodiment; FIG. 30 illustrates actual p values in two-tailed unpaired t-test, in one embodiment;

FIG. 31 illustrates primary and secondary antibodies, in another embodiment;

FIG. 32 illustrates a yield of neurulation implantation, in yet another embodiment;

FIGs. 33A-33B show technical innovations critical to soft and stretchable PFPE- DMA mesh electronics, according to some embodiments;

FIGs. 34A-34C show long-term rearing of cyborg tadpoles to cyborg frogs, according to some embodiments;

FIG. 35 shows immuno staining images depicting the contact between Xenopus brain tissues and mesh electronics, in some embodiments;

FIGs. 36A-36B show procedures for quantitative analysis of fluorescence images, according to some embodiments;

FIG. 37 shows the design of the stretchable mesh electronics with a 32-channel mesh electrode array, according to some embodiments;

FIG. 38A-38F show implantation of stretchable mesh electronics in mouse embryos, according to some embodiments;

FIGs. 39A-39J show implantation of stretchable mesh electronics in neonatal rat brain. FIG. 39A-39B are photographic images showing mesh electronics before (FIG. 39A) and after (FIG. 39B) implantation into a neonatal rat brain; according to some embodiments;

FIGs. 39C-39D are photographic images showing the neonatal rat after stereotactic surgery (FIG. 39C) and after recovering from anesthetics (FIG. 39D), according to some embodiments;

FIG. 39E shows representative filtered voltage traces (300-3,000 Hz bandpass filter), according to some embodiments;

FIG. 39F shows zoomed-in views of the signals highlighted by blue-, green- and red- dashed box-highlighted regions in (FIG. 39E), according to some embodiments;

FIG. 39G shows a raster plot of single-unit action potentials sorted from the recording, according to some embodiments;

FIGs. 39H-39J show principal component analysis (PCA) (FIG. 39H), average waveforms (mean ± s.d.) (FIG. 391) and ISI (FIG. 39J) of two representative units, according to some embodiments;

FIGs. 40A-40H show neural recording in awake cyborg tadpole, according to some embodiments;

FIGs. 41A-41B show the actual p values in two-tailed unpaired t-tests, according to some embodiments; and FIG. 42 shows yield of neurulation implantation.

DETAILED DESCRIPTION

Certain aspects of the present disclosure are generally to systems and methods for implanting a stretchable electronic component into a biological structure, such as tissues, organoids, organs, organisms, and the like. In some cases, the stretchable electronic component is implanted into the tissue of an organism.

The organism may be of any suitable age, for example, a juvenile, an adult, or the like. In some cases, the juvenile may be less than 10 years old, less than 5 years old, less than 3 years old, less thanl year old, less than 4 weeks old, less than 3 weeks old, less than 2 weeks old, less than 1 week old, etc. In some cases, the juvenile organism is one that is prepuberty. For example, the juvenile organism may be one that does not yet exhibit any evidence of maturation of its reproductive organs.

Implantation of a component such as is described herein may be difficult in certain embodiments, as the component may need to conform to the tissues within the organism. In contrast, implantation of such devices to smaller biological structures (for example, embryos) do not require such conformation. As a non-limiting example, if the component was to be implanted into the brain (or other organ) of an organism (for example, a juvenile or an adult organism), the device may be stretchable in some embodiments, e.g., to allow the component to penetrate into the brain. Non-limiting examples of organs include the brain, heart, kidney, or the like.

In some cases, the component may be stretchable. For example, the component may be configured to return (at least partially) to its original structure prior to stretching. For instance, the component may return at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% (perfectly elastic) back to its original structure, measured from when stretching of the component is stopped. Thus, for example, a 1 cm component stretched to 2 cm experiences a 100% stretch in a linear direction, and if it afterwards contracts to 1.5 cm, it exhibits a 50% recovery to its original structure (returning 0.5 cm from its stretch of 1 cm). However, it should be understood that in some embodiments, the component is not stretchable. The stretchability of a component may be based on the composition of the component, and/or the shape of the component. For example, in some embodiments, the component may have a shape and/or may be formed from materials that allow the component to be manipulated or distorted without disrupting their connections, e.g., during stretching, compression, folding, or the like. For example, the component may have an “S” or a serpentine shape.

In some embodiments, the component may be an electronic component that defines at least a portion of an electrical circuit, or portions thereof. Electrical circuits are described in more detail herein, and the electrical circuit may be a self-contained electrical circuit, and/or a portion of the electrical circuit may be interfaceable or connectable with an external electrical device, such as a computer, e.g., using a suitable connector, such as a cable.

In some cases, electronic signals may be determined from the electrical circuit over relatively long periods of time, e.g., where the implant is present within the tissue or organism (or other biological structure) during those times. For instance, the implant may be implanted in an organism for at least 1 day, at least 2 days, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 24 weeks, at least 1 year, at least 2 years, at least 3 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years, at least 50 years, etc. In some cases, the implant may be implanted for a period of time that is at least 5% of the average lifetime of the species to which the organism belongs, and in some cases, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, etc. of the average lifetime. In some cases, the implant is permanent, i.e, the implant remains in the organism until the organism dies.

In certain aspects, e.g., due to the stretchable nature of the device, or a component within the device, a guide may be used to assist in the implantation of the device. For instance, a guide may be used to facilitate entry of the device into the tissue, then the guide removed in some fashion, without removing the device, thereby leaving the device implanted within the tissue. For example, the guide may be physically removed, or allowed to remain in place but dissolved or chemically or biologically removed in some fashion. For instance, the guide may be formed from materials such as a hydrogel or a biodegradable polymer (for example, polycaprolactone, polylactic acid, poly glycolic acid, or the like). Combinations of these and/or other materials are also possible. In addition, in some cases, the guide may be frozen or cooled, e.g., below a melting or glass transition temperature, to enhance its stiffness. Upon insertion, the guide may be heated (e.g., by the subject’s body heat) to a warmer temperature.

The guide may have any suitable shape. For example, the guide may be substantially planar, and the device attached to the guide (e.g., to a surface of the guide), or the guide may be hollow, e.g., forming a tube through which the device can be inserted into the tissue.

One aspect of the present disclosure is now described with reference to FIG 1. In this figure, a device is shown, some or all of which may be stretchable. The device, for example, may comprise components such as a plurality of nodes connected by various interconnects, e.g., forming a mesh structure, where the interconnects have a shape and/or are formed from materials that allow the interconnects to be manipulated or distorted without disrupting their connections, e.g., during stretching, compression, folding, or the like. For example, the interconnects may have an “S” or a serpentine shape (e.g., FIG. 2). In some cases, the interconnects may be formed from materials that can be manipulated by the cells within the component. For example, the cells may be part of a tissue of a subject, for example, the brain, heart, kidney, or the like.

Accordingly, in some cases, a device containing one or more components may be present within a brain or other organ, which may be partially or completely embedded therein. In some cases, the growing brain is able to manipulate or distort the component as part of the growth process, e.g., to stretch the component, and/or to cause the component to form a more suitable embedded shape within the brain, etc. For example, as the brain grows, the cells may cause the component to stretch in various configurations. In some cases, the device may be introduced to a juvenile organism.

In certain embodiments, the device may contain components, including nanoelectric components, that may form electrical circuits, or portions thereof. For instance, some or all of the interconnects may contain metal or other conductive pathways, and/or there may be sensors, stimulators, nanoscale wires, or the like within the device. In some cases, the device may define a self-contained electrical circuit, and/or a portion of the device may be interfaceable or connectable with an external electrical device, such as a computer, using a suitable connector, such as a cable.

In various embodiments, the device within the brain can be connected to electronic circuits extending externally of the brain. The nanoscale wires may form an integral part of the brain, and can be determined or controlled, e.g., using an external electrical device. This allows for the creation of new types of functionalized brain, due to the high degree of electronic control. Accordingly, such devices can be determined and/or controlled at high resolutions. In some cases, such devices can be used in a wide variety of novel applications, including applications in tissue engineering, prosthetics, pacemakers, implants, or the like.

In addition, certain embodiments are generally related to nanoscale wires and nanoelectronics, which in some aspects may be embedded in biological structures, such as tissues, organoids, organs, organisms, and the like. In one embodiment, the biological structure is a neural plate of an embryo, which may develop to form the brain of the growing organism.

Certain aspects are generally directed to flexible or stretchable components that can be used within such devices, which can be implanted into biological structures such as organoids, tissues, or even organisms. For example, the biological structure may be the brain of an organism. The device may comprise biocompatible and/or biodegradable materials, and may in some embodiments also contain growth factors such as growth hormones, extracellular matrix proteins, specific metabolites or nutrients, or the like. In some cases, the device may be porous, e.g., to facilitate cell seeding therein, and/or diffusion into and out of the device, for example, of nutrients, waste products, etc.

In one set of embodiments, the device may include one or more components that may have a shape and/or may be formed from one or more materials that allow the device, or a portion of the device, to be flexible or stretchable. For example, the components may be formed of shapes, such as serpentine shapes, that can be extended. In some cases, the components may not be straight, and can be extended, e.g., when pulled on. For instance, the components may comprise one or more nodes that are connected by various interconnects, e.g., forming a mesh or a network. The nodes may be evenly or nonevenly distributed within the component, and the interconnects may connect them in a regular pattern (for example, in rectangular or triangular arrays of nodes), or in an irregular pattern. As a non-limiting example, FIG. 1, panel I shows a mesh of nodes (dots) in a square array connected by a plurality of interconnects between pairs of nodes (shown as wiggly lines). The nodes may represent points of connectivity, or there may be one or more components at some or all of the nodes, such as conductive pathways, nanoscale wires, sensors, or the like. The same or different components may independently be present at different nodes within a mesh or network. The interconnects connecting two (or more) nodes together may have the same or different shapes or structure within a mesh or network, and different interconnects within the mesh may independently have the same or different shapes. In some cases, an interconnect may have a shape that is extendible. For example, an interconnect may have a straight-line or linear shape, or have shapes that are non-linear, such as S shapes, serpentine shapes (e.g., having two, three, four, or more bends or inflection points), zigzag shapes (e.g., having two, three, four, or more vertices), coiled shapes, or the like. Such interconnect shapes may allow various manipulations to occur without disrupting the connection of the interconnect to the nodes, e.g., during stretching, compression, folding, etc.

In one set of embodiments, an interconnect may comprise one or metal leads and one or more polymers, such as those discussed below. The polymers can include photoresist polymers (such as SU-8), and/or biocompatible polymers (such as Matrigel™). Other examples of photoresist polymers include, but are not limited to, those described below.

In some cases, the device may have an overall filling ratio or area of less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, etc. The filling ratio or area is the area of the physical components of the device, compared to the overall area of the device (including void spaces). Thus, this is a measure of the “porosity” in two dimensions of the device. For example, in some cases, the device may have a mesh structure or layout as described above, where the mesh is relatively open. Devices with smaller filling ratios thus would have greater “open space,” for example, to allow cells to penetrate.

In some cases, the device can be defined by one or more pores. Pores that are too small can hinder or restrict cell access. Thus, in some embodiments, the device may have an average pore size of at least about 100 micrometers, at least about 200 micrometers, at least about 300 micrometers, at least about 400 micrometers, at least about 500 micrometers, at least about 600 micrometers, at least about 700 micrometers, at least about 800 micrometers, at least about 900 micrometers, or at least about 1 mm. However, in other embodiments, pores that are too big may prevent cells from being able to satisfactorily use or even access the pore volume. Thus, in some cases, the device may have an average pore size of no more than about 1.5 mm, no more than about 1.4 mm, no more than about 1.3 mm, no more than about 1.2 mm, no more than about 1.1 mm, no more than about 1 mm, no more than about 900 micrometers, no more than about 800 micrometers, no more than about 700 micrometers, no more than about 600 micrometers, or no more than about 500 micrometers. Combinations of these are also possible, e.g., in one embodiment, the average pore size is at least about 100 micrometers and no more than about 1.5 mm. In addition, larger or smaller pores than these can also be used in a device in certain cases. Pore sizes may be determined using any suitable technique, e.g., through visual inspection, BET measurements, or the like.

In some cases, an interconnect may have a smallest dimension or a maximum cross- sectional dimension of less than about 100 micrometers, less than about 50 micrometers, less than about 40 micrometers, less than about 30 micrometers, less than about 20 micrometers, less than about 10 micrometers, less than about 5 micrometers, less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 80 nm, less than about 50 nm, less than about 30 nm, less than about 10 nm, less than about 5 nm, less than about 2 nm, etc. An interconnect may also have any suitable cross-sectional shape, e.g., circular, square, rectangular, polygonal, elliptical, regular, irregular, etc.

In addition, in one set of embodiments, one or more components within the device are stretchable or flexible. For example, in some cases, a device may comprise a mesh or portions thereof (e.g., interconnects) that can be stretchable or flexible, or can be manipulated or distorted in some fashion. It should be understood that the flexibility of a material is not purely an intrinsic material propriety; a thinner piece of material may offer more flexibility than a comparably thicker piece of the same material. In addition, in some cases, the flexibility of the material may also be a function of its shape, e.g., as discussed above.

In certain instances, a device may have components, such as interconnects, that are sufficiently flexible or stretchable such that the device (or a component thereof, such as an interconnect) may be stretchable in a linear direction by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, etc., for example, before catastrophic failure of the device, breakage, disruption of the connection of the interconnect to the nodes, loss of electrical connections, or the like.

In addition, in certain cases, the device may also exhibit some degree of elasticity, e.g., such that the device may return (at least partially) to its original structure prior to stretching. For instance, the device (or a component thereof, such as an interconnect) may return at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% (perfectly elastic) back to its original structure, measured from when stretching of the material is stopped. Thus, for example, a 1 cm material stretched to 2 cm experiences a 100% stretch in a linear direction, and if it afterwards contracts to 1.5 cm, it exhibits a 50% recovery to its original structure (returning 0.5 cm from its stretch of 1 cm). However, it should be understood that in some embodiments, the device is not elastic.

In some embodiments, the device may have components, such as interconnects, that are sufficiently flexible or stretchable such that the device (or a component thereof, such as an interconnect) may have an effective bending stiffness of at least 0.01 n-Nm, at least 0.02 n-Nm, at least 0.03 n-Nm, at least 0.04 n-Nm, at least 0.05 n-Nm, at least 0.06 n-Nm, at least 0.07 n-Nm, at least 0.08 n-Nm, at least 0.09 n-Nm, at least 0.1 n-Nm, at least 0.2 n-Nm, at least 0.3 n-Nm, at least 0.4 n-Nm, at least 0.5 n-Nm, at least 0.7 n-Nm, at least 1 n-Nm, at least 1.5 n-Nm, at least 2 n-Nm, at least 2.5 n-Nm, at least 3 n-Nm, at least 3.5 n-Nm, at least 4 n-Nm, at least 4.5 n-Nm, at least 5 n-Nm, etc. In some embodiments, the interconnects may have an effective bending stiffness of less than 5 n-Nm, less than 4.5 n-Nm, less than 4 n-Nm, less than 3.5 n-Nm, less than 3 n-Nm, less than 2.5 n-Nm, less than 2 n-Nm, less than 1.9 n-Nm, less than 1.8 n-Nm, less than 1.5 n-Nm, less than 1.3 n-Nm, less than 1 n-Nm, less than 0.9 n-Nm, less than 0.8 n-Nm, less than 0.5 n-Nm, less than 0.3 n-Nm, etc. Combinations of any of these are also possible; for example, the device or interconnect may exhibit an effective bending stiffness of between 0.090 n-Nm and 1.9 n-Nm. See the examples below for an example of determining effective bending stiffness of a material.

In certain embodiments, the device may have components, such as interconnects, that are sufficiently flexible or stretchable such that the device (or a component thereof, such as an interconnect) can be compressed in a linear direction by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, etc., without catastrophic failure of the device, breakage, disruption of the connection of the interconnect to the nodes, loss of electrical connections, or the like.

In addition, in certain embodiments, the device may have components, such as interconnects, that are sufficiently flexible or stretchable such that the device (or a component thereof, such as an interconnect) is foldable by at least 30°, at least 45°, at least 90°, at least 135°, at least 150°, at least 180°, etc. from an initial planar structure.

The device may comprise a variety of materials in different embodiments. For example, the device, or a component thereof, may comprise one or more polymers, such as photoresists, that define interconnects or other components within the device. In some cases, one or more portions of the device may comprise components, such as nanoelectric components, that may form electrical circuits within the device. For example, the device may contain metal or other conductive pathways, e.g., which define an electrical circuit, and/or can be connected to an external electrical device.

Thus, in certain embodiments, the device, or a component thereof, may contain one or more polymers, e.g., photoresists, biocompatible polymers, biodegradable polymers, etc., as is discussed herein. For example, in various embodiments, one or more of the polymers may be a photoresist. While not commonly used in such devices, photoresists are typically used in lithographic techniques, which can be used as discussed herein. For example, the photoresist may be chosen for its ability to react to light to become substantially insoluble (or substantially soluble, in some cases) to a photoresist developer.

Photoresists that can be used include, but are not limited to, SU-8, SI 805, LOR 3A, poly(methyl methacrylate), poly(methyl glutarimide), phenol formaldehyde resin (diazonaphthoquinone/novolac), diazonaphthoquinone (DNQ), Hoechst AZ 4620, Hoechst AZ 4562, Shipley 1400-17, Shipley 1400-27, Shipley 1400-37, or the like. These and many other photoresists are available commercially. Other examples of photoresist polymers include, but are not limited to, those described below, and those described in Int. Pat. Apl. Pub. No. WO 2019/084498, incorporated herein by reference. In some cases, the photoresist may be a soft material, for example, a hydrogel. In some embodiments, the photoresist comprises a polymer formed by photo-curing a fluorinated monomer including cross -linkable function groups using a photoinitiator. One example of such a polymer is perfluoropolyether dimethacrylate (PFPE-DMA). Other examples are discussed in more detail below.

In some cases, one or more of the polymers may be biocompatible and/or biodegradable. Examples of such biocompatible and/or biodegradable polymers include, but are not limited to, poly(lactic-co-glycolic acid), polylactic acid, polyglycolic acid, poly(methyl methacrylate), poly(trimethylene carbonate), collagen, fibrin, polysaccharidic materials such as chitosan or glycosaminoglycans, hyaluronic acid, polycaprolactone, and the like. Certain photoresists are also biocompatible and/or biodegradable in some cases.

Typically, a biocompatible material is one that does not illicit an immune response, or elicits a relatively low immune response, e.g., one that does not impair the device or the tissue it is implanted in from continuing to function for its intended use. In some embodiments, the biocompatible material is able to perform its desired function without eliciting any undesirable local or systemic effects in a subject, e.g., when present within a subject. In some cases, the material is present without eliciting any undesirable local or systemic effects, or such that any biological response by the subject does not substantially affect the ability of the material from continuing to function for its intended use. For example, the device may be able to support appropriate cellular or tissue activity when implanted within a subject, e.g., including the facilitation of molecular and/or mechanical signaling systems, without substantially eliciting undesirable effects in those cells, or undesirable local or systemic responses, or without eliciting a response that causes the device to cease functioning for its intended use.

A biodegradable material typically degrades over time when exposed to a biological system, e.g., through oxidation, hydrolysis, enzymatic attack, phagocytosis, or the like. For example, a biodegradable material can degrade over time when exposed to water (e.g., hydrolysis) or enzymes. In some cases, a biodegradable material is one that exhibits degradation (e.g., loss of mass and/or structure) when exposed to physiological conditions for at least about a month, at least about 6 months, or at least about a year. For example, the biodegradable material may exhibit a loss of mass of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In certain cases, some or all of the degradation products may be resorbed or metabolized, e.g., into cells or tissues. For example, certain biodegradable materials, during degradation, release substances that can be metabolized by cells or tissues.

In some embodiments, the device, or a component thereof, may also contain other materials in addition to the polymers described herein. Non-limiting examples include other polymers, growth hormones, extracellular matrix protein, specific metabolites or nutrients, or the like. For example, in one of embodiments, one or more agents able to promote cell growth can be added to the device, e.g., hormones such as growth hormones, extracellular matrix protein, pharmaceutical agents, vitamins, or the like. Many such growth hormones are commercially available, and may be readily selected by those of ordinary skill in the art based on the specific type of cell or tissue used or desired. Similarly, non-limiting examples of extracellular matrix proteins include gelatin, laminin, fibronectin, heparan sulfate, proteoglycans, entactin, hyaluronic acid, collagen, elastin, chondroitin sulfate, keratan sulfate, Matrigel™, or the like. Many such extracellular matrix proteins are available commercially, and also can be readily identified by those of ordinary skill in the art based on the specific type of cell or tissue used or desired.

As another example, in some embodiments, additional materials can be added to a device, or a component thereof, e.g., to control the size of pores within the device, to promote cell adhesion or growth within the device, to increase the structural stability of the device, to control the flexibility of the device, etc. For instance, additional fibers or other suitable polymers may be added to the device, e.g., electrospun fibers or the like. The additional materials can be formed from any of the materials described herein in reference to devices, e.g., photoresists or biocompatible and/or biodegradable polymers, or other polymers described herein. As another non-limiting example, a glue such as a silicone elastomer glue can be used to control the shape of the device, or a component thereof.

In one set of embodiments, the device, or a component thereof, may contain metal or other conductive pathways, e.g., within interconnects or nodes within the device. Examples of metals for metal leads or pathways that can be used include, but are not limited to platinum, aluminum, gold, silver, copper, molybdenum, tantalum, titanium, nickel, tungsten, chromium, palladium, or the like, as well as any combinations of these and/or other metals. Other examples include conductive polymers such as poly (3, 4-ethylenedioxy thiophene) (PEDOT), poly acetylene, polyphenylene vinylene, polypyrrole, poly thiophene (for example poly (3, 4-ethylenedioxy thiophene)), polyphenylene sulfide, etc.

In some cases, the material can be chosen to be one that is readily introduced into the device, or a component thereof, e.g., using techniques compatible with lithographic techniques. For example, in one set of embodiments, lithographic techniques such as e-beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc. may be used to layer or deposit one or more metals on a substrate. Additional processing steps can also be used to define or register the pathways in some cases.

In some embodiments, more than one metal can be used within a pathway. For example, two, three, or more metals may be used within a pathway. The metals may be deposited in different regions or alloyed together, or in some cases, the metals may be layered on top of each other, e.g., layered on top of each other using various lithographic techniques. If dissimilar metals are layered on top of each other, they may be layered in some embodiments in a “stressed” configuration (although in other embodiments, they may not necessarily be stressed). For example, a chromium/palladium/chromium deposition process, in some embodiments, may form a pre- stressed arrangement that is able to spontaneously form a 3-dimensional structure after release from the substrate. See, e.g., U.S. Pat. Nos. 9,457,128 or 9,786,850, each incorporated herein by reference in its entirety.

In some embodiments, the conductive pathway may be relatively narrow. For example, the conductive pathway may have a smallest dimension or a largest cross-sectional dimension of less than about 5 micrometers, less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 80 nm, less than about 50 nm, less than about 30 nm, less than about 10 nm, less than about 5 nm, less than about 2 nm, etc. The conductive pathway may have any suitable cross-sectional shape, e.g., circular, square, rectangular, polygonal, elliptical, regular, irregular, etc. As is discussed in detail below, such conductive pathways may be achieved using lithographic or other techniques.

In some cases, the conductive pathways may define an electrical circuit that is internally contained within the device (or a component thereof), and/or that extends externally of the device, e.g., such that the electrical circuit is in electrical communication with an external electrical system, such as a computer or a transmitter (for instance, a radio transmitter, a wireless transmitter, an Internet connection, etc.). The device, in some embodiments, may contain components such as nanoelectric components. Non-limiting examples of such components include nanoscale wires, sensors such as nanosensors, transistors such as field effect transistors, resistors, capacitors, inductors, diodes, integrated circuits, batteries, power sources, RFID tags, antennae, transmitter, or the like, which may be present in one or more electrical circuit within the device.

In addition, in certain cases, a component within the devicemay comprise an electrode. The electrode may comprise any suitable material, for example, carbon, or metals such as gold, platinum, silver, or the like. In some cases, the electrode may be used to determine a property of the device (e.g., an electrical property, a chemical property, a mechanical property, etc.), and/or to apply a stimulus (e.g., an electrical stimulus) to the biological structure. In some cases, a conductive polymer may also be used with the electrode. Non-limiting examples of conductive polymers include poly(3,4- ethylenedioxythiophene) (PEDOT), polyacetylene, polyphenylene vinylene, polypyrrole, poly thiophene (for example poly (3, 4-ethylenedioxy thiophene)), polyphenylene sulfide, or other conductive polymers such as those described herein.

In some cases, the components may be used to determine a property of the device, e.g., when it is embedded within a biological structure, such as discussed herein. For example, one or more locations within a device (for example, an electrode or a nanoscale wire, etc.) may be determined to determine a property, such as a chemical property, an electrical property, a mechanical property, or the like. Other examples include sensing Ca²⁺ spikes, voltage changes, cell signaling pathways, ion concentrations, pH changes, sensing of biomolecules or reaction entities, etc. In some cases, the locations are defined as one or more nodes within the device, some or all of which may be individually addressable. For example, a node within a devicemay comprise a nanoscale wire, such as those discussed in more detail below.

In certain embodiments, the conductive pathways may define an electrical circuit that is interfaceable or connectable with an external electrical device, such as a computer, using a suitable connector. For example, the device may be directly connected to an external device (for instance, using an interface such as described in U.S. Pat. Apl. Pub. No. 2018/0328884, incorporated herein by reference in its entirety). In some cases, a suitable connector, such as a cable, may be used to make such a connection between an electrical circuit within a device and the external device. Non-limiting examples of cables include those commercially available, such as ribbon cables, flexible flat cable, 8-pin cables, 16-pin cables, etc., or other electrical cables. However, an external connection is not always required, and in some cases, the device may be or contain a self-contained electrical circuit. For example, the circuit may be able to transmit information wirelessly to an external device, store information for later access (e.g., after sacrificing the organoids, organs, or organisms), or the like.

In addition, in certain embodiments, the device, or a component thereof, may be able to communicate with an external device using wireless communications, e.g., in addition to and/or instead of an electrical connection. For example, the device, or a component thereof, may contain a transmitter (for instance, a radio transmitter, a wireless transmitter, an Internet connection, etc.) and/or a receiver, e.g., which may be in communication with a transmitter and/or a receiver on an external device.

In some embodiments, more than one electrical circuit and/or more than one conductive pathway may be used within a device, or a component thereof. For example, multiple conductive pathways or circuits can be used such that some or all of the nodes may be individually electronically addressable within the device. However, in other embodiments, more than one node may be addressable by a particular conductive pathway.

In various aspects, cells may be cultured on a device and allowed to grow to become a biological structure, such as an organoid or a tissue, or the device may be exposed to a preexisting biological structure, such as a tissue. The device can become partially or completely embedded within the structure, e.g., during growth of biological structure. The biological structure may be, for example, tissues, organoids, organs, organisms, and the like.

A variety of cells may be used with the device in some embodiments. The cell may be an isolated cell, a cell aggregate, in a tissue construct containing cells, or the like. Examples of cells include, but are not limited to, a bacterium or other single-cell organism, or a eukaryotic cell, such as a plant cell, or an animal cell. If the cell is from a multicellular organism, the cell may be from any part of the organism. For instance, if the cell is from an animal, the cell may be a cardiac cell, a fibroblast, a keratinocyte, a hepatocyte, a chondrocyte, a neural cell, an osteocyte, an osteoblast, a muscle cell, a blood cell, an endothelial cell, an immune cell (e.g., a T-cell, a B-cell, a macrophage, a neutrophil, a basophil, a mast cell, an eosinophil), etc. In some cases, the cells may be cancer cells.

Examples of cells able to form suitable organoid or organs include, but are not limited to brain cells, cardiac (heart) cells, nephron (kidney) cells, or the like. In some cases, the cells are cancer cells, e.g., that can grow to form a tumor. In certain embodiments, the cells are stem cells, e.g., pluripotent stem cells. In some cases, the cells may also be exposed to other compounds, such as drugs, to determine their effects on the growth of the cells into organoids, organs, or organisms. This may be useful, for example, for drug testing.

In some cases, the device may contain at least part of the biological structure. For example, the device may be manipulated or distorted, e.g., by the cells, during growth of the biological structure.

The devicemay be partially or completely embedded within the biological structure. For example, the device may be completely embedded inside of the biological structure, such that no portion of the device is exposed externally of the biological structure. In another set of embodiments, the device is only partially embedded within the biological structure, and at least a portion of the device is exposed externally of the biological structure. For instance, an external portion of the device may be used to electrically connect the device to an external electrical device, such as a computer. For instance, a suitable connector may be connected to the exposed portion of the device, e.g., to form a connection between an electrical circuit within a device, and the external device.

In some cases, the device contains a relatively large part of the biological structure. For instance, the device may contain at least 30 vol%, at least 40 vol%, at least 50 vol%, at least 60 vol%, at least 70 vol%, at least 80 vol%, at least 90 vol%, or at least 95 vol% of the biological structure. In addition, in some cases, the device may be distributed within a relatively large part of the biological structure. For example, the device may be manipulated or distorted such that at least 10 vol% of the biological structure is no more than 5, 10, or 30 micrometers from the device embedded therein, and in some cases, at least 20 vol%, at least 30 vol%, at least 40 vol%, at least 50 vol%, at least 60 vol%, at least 70 vol%, at least 80 vol%, at least 90 vol%, or at least 95 vol% of the biological structure is no more than 5, 10, or 30 micrometers from the device. In some cases, the device may be manipulated or distorted, e.g., by the tissue, to stretch the device. For example, the biological structure may grow and or expand, and the embedded device may be stretched along with the biological structure as it expands. In certain embodiments, the device may exhibit a lower filling ratio as it is expanded by the biological structure. For instance, the device may have an first, initial filling ratio (e.g., prior to adding cells) of less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, etc., and a second filling ratio, after expansion, that is less than the initial filling ratio. For example, the second filling ratio may be less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the initial filing ratio. In some cases, the second filing ratio may be less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, etc. In addition, in some cases, the cells may case strain to the device during expansion. For example, at least a portion of the device may exhibit a tensile strain of at least 10% or at least 20%.

A variety of biological structures are contemplated, such as tissues, organoids, organs, whole organisms, or the like. Non-limiting examples of tissues or organs include brain, heart, a kidney, etc. In some cases, the tissue or organ may also arise from cancerous or tumor cells.

In addition, in some cases, the biological structure may be an organism, i.e., an entire organism. The organism may be any suitable organism, including plants or animals, such as invertebrate or vertebrate organisms. The organism may be, for example, an invertebrate (e.g., a fruit fly), a fish (e.g., a zebrafish), an amphibian (e.g., a frog), a reptile, a bird, or a human or non-human mammal, such as a monkey, a cow, a sheep, a goat, a horse, a rabbit, a pig, a rodent such as a mouse or a rat, a dog, or a cat.

In addition, in one set of embodiments, a device (with or without a biological structure) may be implanted into an organism. For example, an organoid or an organ containing a device can be implanted within an organism. The organism may be a human or non-human mammal, such as a monkey, cow, sheep, goat, horse, rabbit, pig, mouse, rat, dog, or cat. The organoid or organ may be from the same or different species as the organism, and may be from the same individual or a different one.

Certain aspects of the present disclosure are generally directed to systems and methods for preparing devices such as those described herein. The devices can be fabricated, for example, using well-known lithographic techniques such as those discussed below. In various embodiments, a device is constructed by assembling various polymers, metals, and other components (for example, nanoscale wires) together on a substrate. For example, lithographic techniques such as e-beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc. may be used to pattern polymers, metals, etc. on the substrate. After assembly, at least a portion of the substrate (e.g., a sacrificial material) may be removed, allowing the device to be partially or completely removed from the substrate. Other materials may also be added to the device, e.g., to help stabilize the structure, to add additional agents to enhance its biocompatibility, etc. The devicecan be used in vivo, e.g., by implanting it in a subject, and/or in vitro, e.g., by seeding cells, etc. on the device. In addition, in some cases, cells may initially be grown or cultured on the device, e.g., to form a biological structure, such as tissues, organoids, organs, organisms, and the like. In some cases, as discussed, the device may be sufficiently flexible such that the device becomes embedded within the biological structures as it forms.

For example, in one set of embodiments, a device may be constructed by providing a substrate, depositing a sacrificial layer on the substrate, then patterning a first photoresist on the sacrificial layer, a conductive pathway on the first photoresist, and a second photoresist on the conductive pathway, and removing the sacrificial layer to produce the device. See, e.g., FIG. 3. The first and second photoresists may comprise the same or different materials. Optionally, other components can also be added to the device, before or during formation, such as electrode components, nanoscale wires, connectors such as cables, or the like.

The substrate may be chosen to be one that can be used for lithographic techniques such as e-beam lithography or photolithography, or other lithographic techniques including those discussed herein. For example, the substrate may comprise or consist essentially of a semiconductor material such as silicon, although other substrate materials (e.g., a metal) can also be used. Typically, the substrate is one that is substantially planar, e.g., so that polymers, metals, and the like can be patterned on the substrate. In some cases, a portion of the substrate can be oxidized, e.g., forming SiCh and/or ShN4 on a portion of the substrate, which may facilitate subsequent addition of materials (metals, polymers, etc.) to the substrate.

In certain embodiments, one or more polymers can also be deposited or otherwise formed prior to depositing the sacrificial material. In some cases, the polymers may be deposited or otherwise formed as a layer of material on the substrate. Deposition may be performed using any suitable technique, e.g., using lithographic techniques such as e-beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc. In some cases, some or all of the polymers may be biocompatible and/or biodegradable. The polymers that are deposited may also comprise methyl methacrylate and/or poly (methyl methacrylate), in some embodiments.

Next, a sacrificial material may be deposited. The sacrificial material can be chosen to be one that can be removed without substantially altering other materials (e.g., polymers, other metals, nanoscale wires, etc.) deposited thereon. For example, in one embodiment, the sacrificial material may be a metal, e.g., one that is easily etchable. For instance, the sacrificial material can comprise germanium or nickel, which can be etched or otherwise removed, for example, using a peroxide (e.g., H2O2) or a nickel etchant (many of which are readily available commercially). In some cases, the sacrificial material may be deposited on oxidized portions or polymers previously deposited on the substrate. In some cases, the sacrificial material is deposited as a layer. The layer can have a thickness of less than about 5 micrometers, less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, etc.

In some embodiments, a first photoresist can be deposited, e.g., on the sacrificial material. The photoresist may include one or more polymers, which may be deposited as one or more layers. Examples of photoresist include, but are not limited to, SU-8, SI 805, LOR 3A, poly(methyl methacrylate), poly(methyl glutarimide), phenol formaldehyde resin (diazonaphthoquinone/novolac), diazonaphthoquinone (DNQ), Hoechst AZ 4620, Hoechst AZ 4562, Shipley 1400-17, Shipley 1400-27, Shipley 1400-37, etc., as well as any others discussed herein.

The photoresist can be used to at least partially define a device. In one set of embodiments, the photoresist may be deposited as a layer of material, such that portions of the photoresist may be subsequently removed. For example, the photoresist can be deposited using lithographic techniques such as e-beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc., or using other techniques for removing polymer that are known to those of ordinary skill in the art. In some cases, more than one photoresist is used, e.g., deposited as more than one layer (e.g., sequentially), and each layer may independently have a thickness of less than about 5 micrometers, less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, etc. For example, in some embodiments, portions of the photoresist may be exposed to light (visible, UV, etc.), electrons, ions, X-rays, etc. (e.g., projected onto the photoresist), and the exposed portions can be etched away (e.g., using suitable etchants, plasma, etc.) to produce the pattern.

Accordingly, the photoresist may be formed into a particular pattern, e.g., in a grid or a mesh, e.g., as discussed herein. For instance, the pattern may include a mesh and interconnects that have a shape that allow the interconnects to be manipulated or distorted without disrupting their connections, e.g., during stretching, compression, folding, or the like. The pattern can be regular or irregular.

Next, a metal or other conductive material can be deposited e.g., on one of the previous materials, to form conductive pathways within the device. More than one metal can be used, which may be deposited as one or more layers. For example, a first metal may be deposited, and a second metal may be deposited on at least a portion of the first metal. Optionally, more metals can be used, e.g., a third metal may be deposited on at least a portion of the second metal, and the third metal may be the same or different from the first metal. In some cases, each metal may independently have a thickness of less than about 5 micrometers, less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 80 nm, less than about 60 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, less than about 10 nm, less than about 8 nm, less than about 6 nm, less than about 4 nm, or less than about 2 nm, etc., and the layers may be of the same or different thicknesses.

Any suitable technique can be used for depositing metals, and if more than one metal is used, the techniques for depositing each of the metals may independently be the same or different. For example, in one set of embodiments, deposition techniques such as sputtering can be used. Other examples include, but are not limited to, physical vapor deposition, vacuum deposition, chemical vapor deposition, cathodic arc deposition, evaporative deposition, e-beam PVD, pulsed laser deposition, ion-beam sputtering, reactive sputtering, ion-assisted deposition, high-target-utilization sputtering, high-power impulse magnetron sputtering, gas flow sputtering, or the like.

The metals can be chosen in some cases such that the deposition process yields a prestressed arrangement, e.g., due to atomic lattice mismatch, which causes the subsequent metal leads to warp or bend, for example, once released from the substrate. Although such processes were typically undesired in the prior art, in certain embodiments of the present disclosure, such pre-stressed arrangements may be used to cause the resulting device to form a 3-dimensional structure, in some cases spontaneously, upon release from the substrate. See, e.g., U.S. Pat. Apl. Pub. Nos. 2014/0073063, 2014/0074253, 2017/0069858, 2017/0072109, each of which is incorporated herein by reference in its entirety. However, it should be understood that in other embodiments, the metals may not necessary be deposited in a prestressed arrangement.

Examples of metals that can be deposited (stressed or unstressed) include, but are not limited to, aluminum, gold, silver, copper, molybdenum, tantalum, titanium, nickel, tungsten, chromium, palladium, as well as any combinations of these and/or other metals. For example, a chromium/gold/chromium deposition process can be used, as is shown in FIG. 3.

In certain embodiments, a second photoresist can be deposited on the previous materials. The second photoresist may be the same or different from the first photoresist, and may include any of the photoresist materials discussed herein, including any of those described with reference to the first photoresist. The second photoresist may include one or more polymers, which may be deposited as one or more layers. In some embodiments, the second photoresist may be deposited on one or more portions of a substrate, e.g., as a layer of material such that portions of the second photoresist can be subsequently removed, e.g., using lithographic techniques such as e-beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc., or using other techniques for removing photoresist that are known to those of ordinary skill in the art. In some cases, more than one photoresist may be used, e.g., deposited as more than one layer (e.g., sequentially), and each layer may independently have a thickness of less than about 5 micrometers, less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, etc.

After formation of the device, some or all of the sacrificial material may then be removed in some cases. In one set of embodiments, for example, at least a portion of the sacrificial material is exposed to an etchant able to remove the sacrificial material. For example, if the sacrificial material is a metal such as nickel, a suitable etchant (for example, a metal etchant such as a nickel etchant, acetone, etc.) can be used to remove the sacrificial metal. Many such etchants may be readily obtained commercially. In addition, in some embodiments, the device can also be dried, e.g., in air (e.g., passively), by using a heat source, by using a critical point dryer, etc.

Other materials may be also added to the device, e.g., before or after it forms a 3- dimensional structure, for example, to help stabilize the structure, to add additional agents to enhance its biocompatibility (e.g., growth hormones, extracellular matrix protein, Matrigel™, etc.), to cause it to form a suitable 3-dimension structure, to control pore sizes, etc. Nonlimiting examples of such materials have been previously discussed above, and include other polymers, growth hormones, extracellular matrix protein, specific metabolites or nutrients, additional materials, or the like.

In addition, in some cases, the device is exposed to cells, which can be cultured or allowed to grow, e.g., to form a biological structure. In some cases, the cells are plated or seeded as individual cells, although in certain cases, larger cell assemblies (tissues, embryos, etc.) may be used. In one set embodiments, the device may be exposed to cells in vitro, and/or the device may be exposed or even submerged within a suitable cell growth medium. Such media are widely available commercially. In some embodiments, the device can be subsequently implanted in vivo into a subject, e.g., upon the growth of tissue, an organ, an organoid, etc. However, it should be understood that implantation is not required in all embodiments, for example, in cases where an entire organism develops from the cells.

In addition, it should be understood that exposure to cells is not necessarily required in all embodiments. For instance, in one set of embodiments, the device may be prepared without the presence of cells. For example, the device may be sold as part of a kit, and the user may expose the device to cells (or use it for other purposes).

In addition, the device can be interfaced in some embodiments with one or more electronics, e.g., an external electrical system such as a computer or a transmitter (for instance, a radio transmitter, a wireless transmitter, etc.), e.g., as discussed herein. The interfacing may occur at any suitable time, e.g., before or after exposure to cells, before or after a biological structure (e.g., an organoid or an organism) has formed, before or after sale to a user, or the like.

For instance, in some cases, electronic testing of the device may be performed. The device, or a portion thereof, can be connected to an external electrical circuit, e.g., to electronically interrogate or otherwise determine the electronic state of the device. For example, the device may comprise one or more nanoscale wires, or other nanoelectronic components, that can be used as sensors. Such determinations may be performed quantitatively and/or qualitatively, depending on the application, and can involve all, or only a portion, of the device, e.g., as discussed herein.

Thus, as mentioned, in some aspects, the device can comprise one or more nanoscale wires. For instance, one or more nodes may contain nanoscale wires, and/or nanoscale wires may be contained within interconnects, or the like. In some cases, the device within the organoids, organs, or organisms may include one or more sensors or stimulators, interconnected with stretchable mesh interconnects, to form a network, e.g., as is shown in FIGS. 1 and 2. The sensors or stimulators may, in some embodiments, comprise nanoscale wires, such as those described herein. Such sensors may be monitored, e.g., individually or collectively.

Non-limiting examples of suitable nanoscale wires include carbon nanotubes, nanorods, nanowires, organic and inorganic conductive and semiconducting polymers, metal nanoscale wires, semiconductor nanoscale wires (for example, formed from silicon), and the like. If carbon nanotubes are used, they may be single-walled and/or multi-walled, and may be metallic and/or semiconducting in nature. Other conductive or semiconducting elements that may not be nanoscale wires, but are of various small nanoscopic-scale dimension, also can be used within the device.

In general, a “nanoscale wire” (also known herein as a “nanoscopic-scale wire” or “nanoscopic wire”) generally is a wire or other nanoscale object, that at any point along its length, has at least one cross-sectional dimension and, in some embodiments, two orthogonal cross-sectional dimensions (e.g., a diameter) of less than 1 micrometer, less than about 500 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 70, less than about 50 nm, less than about 20 nm, less than about 10 nm, less than about 5 nm, than about 2 nm, or less than about 1 nm. In some embodiments, the nanoscale wire is generally cylindrical. In other embodiments, however, other shapes are possible; for example, the nanoscale wire can be faceted, i.e., the nanoscale wire may have a polygonal cross-section. The cross-section of a nanoscale wire can be of any arbitrary shape, including, but not limited to, circular, square, rectangular, annular, polygonal, or elliptical, and may be a regular or an irregular shape. The nanoscale wire can also be solid or hollow.

In some cases, the nanoscale wire has one dimension that is substantially longer than the other dimensions of the nanoscale wire. For example, the nanoscale wire may have a longest dimension that is at least about 1 micrometer, at least about 3 micrometers, at least about 5 micrometers, or at least about 10 micrometers or about 20 micrometers in length, and/or the nanoscale wire may have an aspect ratio (longest dimension to shortest orthogonal dimension) of greater than about 2:1, greater than about 3:1, greater than about 4:1, greater than about 5:1, greater than about 10:1, greater than about 25:1, greater than about 50:1, greater than about 75:1, greater than about 100:1, greater than about 150:1, greater than about 250:1, greater than about 500:1, greater than about 750:1, or greater than about 1000:1 or more in some cases.

In some embodiments, a nanoscale wire is substantially uniform, or the nanowire may have a variation in average diameter of the nanoscale wire of less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. For example, the nanoscale wires may be grown from substantially uniform nanoclusters or particles, e.g., colloid particles. See, e.g., U.S. Patent No. 7,301,199, issued November 27, 2007, entitled “Nanoscale Wires and Related Devices,” by Lieber, et al., incorporated herein by reference in its entirety. In some cases, the nanoscale wire may be one of a population of nanoscale wires having an average variation in diameter, of the population of nanowires, of less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%.

In some embodiments, a nanoscale wire has a conductivity of or of similar magnitude to any semiconductor or any metal. The nanoscale wire can be formed of suitable materials, e.g., semiconductors, metals, etc., as well as any suitable combinations thereof. In some cases, the nanoscale wire will have the ability to pass electrical charge, for example, being electrically conductive. For example, the nanoscale wire may have a relatively low resistivity, e.g., less than about 10'³ Ohm m, less than about 10'⁴ Ohm m, less than about 10'⁶ Ohm m, or less than about 10'⁷ Ohm m. The nanoscale wire can, in some embodiments, have a conductance of at least about 1 microsiemens, at least about 3 microsiemens, at least about 10 microsiemens, at least about 30 microsiemens, or at least about 100 microsiemens.

The nanoscale wire can be solid or hollow, in various embodiments. As used herein, a “nanotube” is a nanoscale wire that is hollow, or that has a hollowed-out core, including those nanotubes known to those of ordinary skill in the art. As another example, a nanotube may be created by creating a core/shell nanowire, then etching away at least a portion of the core to leave behind a hollow shell. Accordingly, in one set of embodiments, the nanoscale wire is a non-carbon nanotube. In contrast, a “nanowire” is a nanoscale wire that is typically solid (i.e., not hollow). Thus, in one set of embodiments, the nanoscale wire may be a semiconductor nanowire, such as a silicon nanowire.

For example, in one embodiment, a nanoscale wire may comprise or consist essentially of a metal. Non-limiting examples of potentially suitable metals include aluminum, gold, silver, copper, molybdenum, tantalum, titanium, nickel, tungsten, chromium, or palladium. In another set of embodiments, a nanoscale wire comprises or consists essentially of a semiconductor. Typically, a semiconductor is an element having semiconductive or semi-metallic properties (i.e., between metallic and non-metallic properties). An example of a semiconductor is silicon. Other non-limiting examples include elemental semiconductors, such as gallium, germanium, diamond (carbon), tin, selenium, tellurium, boron, or phosphorous. In other embodiments, more than one element may be present in the nanoscale wire as the semiconductor, for example, gallium arsenide, gallium nitride, indium phosphide, cadmium selenide, etc. Still other examples include a Group II- VI material (which includes at least one member from Group II of the Periodic Table and at least one member from Group VI, for example, ZnS, ZnSe, ZnSSe, ZnCdS, CdS, or CdSe), or a Group III-V material (which includes at least one member from Group III and at least one member from Group V, for example GaAs, GaP, GaAsP, InAs, InP, AlGaAs, or InAsP).

In certain embodiments, the semiconductor can be undoped or doped (e.g., p-type or n-type). For example, in one set of embodiments, a nanoscale wire may be a p-type semiconductor nanoscale wire or an n-typc semiconductor nanoscale wire, and can be used as a component of a transistor such as a field effect transistor (“FET”). For instance, the nanoscale wire may act as the “gate” of a source-gate-drain arrangement of a FET, while metal leads or other conductive pathways (as discussed herein) are used as the source and drain electrodes.

In some embodiments, a dopant or a semiconductor may include mixtures of Group IV elements, for example, a mixture of silicon and carbon, or a mixture of silicon and germanium. In other embodiments, the dopant or the semiconductor may include a mixture of a Group III and a Group V element, for example, BN, BP, BAs, AIN, A1P, AlAs, AlSb, GaN, GaP, GaAs, GaSb, InN, InP, InAs, or InSb. Mixtures of these may also be used, for example, a mixture of BN/BP/BAs, or BN/A1P. In other embodiments, the dopants may include alloys of Group III and Group V elements. For example, the alloys may include a mixture of AlGaN, GaPAs, InPAs, GalnN, AlGalnN, GalnAsP, or the like. In other embodiments, the dopants may also include a mixture of Group II and Group VI semiconductors. For example, the semiconductor may include ZnO, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, BeS, BeSe, BeTe, MgS, MgSe, or the like. Alloys or mixtures of these dopants are also be possible, for example, (ZnCd)Se, or Zn(SSe), or the like. Additionally, alloys of different groups of semiconductors may also be possible, for example, a combination of a Group II-Group VI and a Group III-Group V semiconductor, for example, (GaAs)_x(ZnS)i-_x. Other examples of dopants may include combinations of Group IV and Group VI elements, such as GeS, GeSe, GeTe, SnS, SnSe, SnTe, PbO, PbS, PbSe, or PbTe. Other semiconductor mixtures may include a combination of a Group I and a Group VII, such as CuF, CuCl, CuBr, Cui, AgF, AgCl, AgBr, Agl, or the like. Other dopant compounds may include different mixtures of these elements, such as BeSiN2, CaCN2, ZnGeP₂, CdSnAs₂, ZnSnSb₂, CuGeP₃, CuSi₂P₃, Si₃N₄, Ge₃N₄, A1₂O₃, (Al, Ga, In)₂(S, Se, Te)₃, AI2CO, (Cu, Ag)(Al, Ga, In, Tl, Fe)(S, Se, Te)2 and the like.

The doping of the semiconductor to produce a p-type or n-typc semiconductor may be achieved via bulk-doping in certain embodiments, although in other embodiments, other doping techniques (such as ion implantation) can be used. Many such doping techniques that can be used will be familiar to those of ordinary skill in the art, including both bulk doping and surface doping techniques. A bulk-doped article (e.g. an article, or a section or region of an article) is an article for which a dopant is incorporated substantially throughout the crystalline lattice of the article, as opposed to an article in which a dopant is only incorporated in particular regions of the crystal lattice at the atomic scale, for example, only on the surface or exterior. For example, some articles are typically doped after the base material is grown, and thus the dopant only extends a finite distance from the surface or exterior into the interior of the crystalline lattice. It should be understood that “bulk-doped” does not define or reflect a concentration or amount of doping in a semiconductor, nor does it necessarily indicate that the doping is uniform. “Heavily doped” and “lightly doped” are terms the meanings of which are clearly understood by those of ordinary skill in the art. In some embodiments, one or more regions comprise a single monolayer of atoms (“deltadoping”). In certain cases, the region may be less than a single monolayer thick (for example, if some of the atoms within the monolayer are absent). As a specific example, the regions may be arranged in a layered structure within the nanoscale wire, and one or more of the regions can be delta-doped or partially delta-doped.

Accordingly, in one set of embodiments, the nanoscale wires may include a heterojunction, e.g., of two regions with dissimilar materials or elements, and/or the same materials or elements but at different ratios or concentrations. The regions of the nanoscale wire may be distinct from each other with minimal cross-contamination, or the composition of the nanoscale wire can vary gradually from one region to the next. The regions may be both longitudinally arranged relative to each other, or radially arranged (e.g., as in a core/shell arrangement) on the nanoscale wire. Each region may be of any size or shape within the wire. The junctions may be, for example, a p/n junction, a p/p junction, an n/n junction, a p/i junction (where i refers to an intrinsic semiconductor), an n/i junction, an i/i junction, or the like. The junction can also be a Schottky junction in some embodiments. The junction may also be, for example, a semiconductor/semiconductor junction, a semiconductor/metal junction, a semiconductor/insulator junction, a metal/metal junction, a metal/insulator junction, an insulator/insulator junction, or the like. The junction may also be a junction of two materials, a doped semiconductor to a doped or an undoped semiconductor, or a junction between regions having different dopant concentrations. The junction can also be a defected region to a perfect single crystal, an amorphous region to a crystal, a crystal to another crystal, an amorphous region to another amorphous region, a defected region to another defected region, an amorphous region to a defected region, or the like. More than two regions may be present, and these regions may have unique compositions or may comprise the same compositions. As one example, a wire can have a first region having a first composition, a second region having a second composition, and a third region having a third composition or the same composition as the first composition. Non-limiting examples of nanoscale wires comprising heterojunctions (including core/shell heterojunctions, longitudinal heterojunctions, etc., as well as combinations thereof) are discussed in U.S. Patent No. 7,301,199, issued November 27, 2007, entitled “Nanoscale Wires and Related Devices,” by Lieber, et al., incorporated herein by reference in its entirety.

In some embodiments, a nanoscale wire is a bent or a kinked nanoscale wire. A kink is typically a relatively sharp transition or turning between a first substantially straight portion of a wire and a second substantially straight portion of a wire. For example, a nanoscale wire may have 1, 2, 3, 4, or 5 or more kinks. In some cases, the nanoscale wire is formed from a single crystal and/or comprises or consists essentially of a single crystallographic orientation, for example, a <110> crystallographic orientation, a <112> crystallographic orientation, or a <1 120> crystallographic orientation. It should be noted that the kinked region need not have the same crystallographic orientation as the rest of the semiconductor nanoscale wire. In some embodiments, a kink in the semiconductor nanoscale wire may be at an angle of about 120° or a multiple thereof. The kinks can be intentionally positioned along the nanoscale wire in some cases. For example, a nanoscale wire may be grown from a catalyst particle by exposing the catalyst particle to various gaseous reactants to cause the formation of one or more kinks within the nanoscale wire. Non-limiting examples of kinked nanoscale wires, and suitable techniques for making such wires, are disclosed in International Patent Application No. PCT/US2010/050199, filed September 24, 2010, entitled “Bent Nanowires and Related Probing of Species,” by Tian, et al., published as WO 2011/038228 on March 31, 2011, incorporated herein by reference in its entirety.

In one set of embodiments, the nanoscale wire is formed from a single crystal, for example, a single crystal nanoscale wire comprising a semiconductor. A single crystal item may be formed via covalent bonding, ionic bonding, or the like, and/or combinations thereof. While such a single crystal item may include defects in the crystal in some cases, the single crystal item is distinguished from an item that includes one or more crystals, not ionically or covalently bonded, but merely in close proximity to one another.

In some embodiments, the nanoscale wires used herein are individual or free-standing nanoscale wires. For example, an “individual” or a “free-standing” nanoscale wire may, at some point in its life, not be attached to another article, for example, with another nanoscale wire, or the free-standing nanoscale wire may be in solution. This is in contrast to nanoscale features etched onto the surface of a substrate, e.g., a silicon wafer, in which the nanoscale features are never removed from the surface of the substrate as a free-standing article. This is also in contrast to conductive portions of articles which differ from surrounding material only by having been altered chemically or physically, in situ, i.e., where a portion of a uniform article is made different from its surroundings by selective doping, etching, etc. An “individual” or a “free-standing” nanoscale wire is one that can be (but need not be) removed from the location where it is made, as an individual article, and transported to a different location and combined with different components to make a functional device such as those described herein and those that would be contemplated by those of ordinary skill in the art upon reading this disclosure.

In various embodiments, more than one nanoscale wire may be present within the device. The nanoscale wires may each independently be the same or different. For example, the device can comprise at least 5 nanoscale wires, at least about 10 nanoscale wires, at least about 30 nanoscale wires, at least about 50 nanoscale wires, at least about 100 nanoscale wires, at least about 300 nanoscale wires, at least about 1000 nanoscale wires, etc. The nanoscale wires may be distributed uniformly or non-uniformly throughout the device. In some cases, the nanoscale wires may be distributed at an average density of at least about 10 nanoscale wires/mm³, at least about 30 nanoscale wires/mm³, at least about 50 nanoscale wires/mm³, at least about 75 nanoscale wires/mm³, or at least about 100 nanoscale wires/mm³. In certain embodiments, the nanoscale wires are distributed within the device such that the average separation between a nanoscale wire and its nearest neighboring nanoscale wire is less than about 2 mm, less than about 1 mm, less than about 500 micrometers, less than about 300 micrometers, less than about 100 micrometers, less than about 50 micrometers, less than about 30 micrometers, or less than about 10 micrometers.

Within the device, some or all of the nanoscale wires may be individually electronically addressable. For instance, in some cases, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or substantially all of the nanoscale wires within the device may be individually electronically addressable. In some embodiments, an electrical property of a nanoscale wire can be individually determinable (e.g., being partially or fully resolvable without also including the electrical properties of other nanoscale wires), and/or such that the electrical property of a nanoscale wire may be individually controlled (e.g., by applying a desired voltage or current to the nanoscale wire, for instance, without simultaneously applying the voltage or current to other nanoscale wires). In other embodiments, however, at least some of the nanoscale wires can be controlled within the same electronic circuit (e.g., by incorporating the nanoscale wires in series and/or in parallel), such that the nanoscale wires can still be electronically controlled and/or determined.

The nanoscale wire, in some embodiments, may be responsive to a property external of the nanoscale wire, e.g., a chemical property, an electrical property, a physical property, etc. Such determination may be qualitative and/or quantitative. For example, in one set of embodiments, the nanoscale wire may be responsive to voltage. For instance, the nanoscale wire may exhibit a voltage sensitivity of at least about 5 microsiemens/V; by determining the conductivity of a nanoscale wire, the voltage surrounding the nanoscale wire may thus be determined. In other embodiments, the voltage sensitivity can be at least about 10 microsiemens/V, at least about 30 microsiemens/V, at least about 50 microsiemens/V, or at least about 100 microsiemens/V. Other examples of electrical properties that can be determined include resistance, resistivity, conductance, conductivity, impendence, or the like.

As another example, a nanoscale wire may be responsive to a chemical property of the environment surrounding the nanoscale wire. For example, an electrical property of the nanoscale wire can be affected by a chemical environment surrounding the nanoscale wire, and the electrical property can be thereby determined to determine the chemical environment surrounding the nanoscale wire. As a specific non-limiting example, the nanoscale wires may be sensitive to pH or hydrogen ions. Further non-limiting examples of such nanoscale wires are discussed in U.S. Patent No. 7,129,554, filed October 31, 2006, entitled “Nanosensors,” by Lieber, et al., incorporated herein by reference in its entirety. As an example, the nano scale wire may have the ability to bind to an analyte indicative of a chemical property of the environment surrounding the nanoscale wire (e.g., hydrogen ions for pH, or concentration for an analyte of interest), and/or the nanoscale wire may be partially or fully functionalized, i.e. comprising surface functional moieties, to which an analyte is able to bind, thereby causing a determinable property change to the nanoscale wire, e.g., a change to the resistivity or impedance of the nanoscale wire. The binding of the analyte can be specific or non-specific. Functional moieties may include simple groups, selected from the groups including, but not limited to, -OH, -CHO, -COOH, -SO3H, -CN, - NH2, -SH, -COSH, -COOR, halide; biomolecular entities including, but not limited to, amino acids, proteins, sugars, DNA, antibodies, antigens, and enzymes; grafted polymer chains with chain length less than the diameter of the nanowire core, selected from a group of polymers including, but not limited to, polyamide, polyester, polyimide, polyacrylic; a shell of material comprising, for example, metals, semiconductors, and insulators, which may be a metallic element, an oxide, an sulfide, a nitride, a selenide, a polymer and a polymer gel.

In some embodiments, a reaction entity may be bound to a surface of the nanoscale wire, and/or positioned in relation to the nanoscale wire such that the analyte can be determined by determining a change in a property of the nanoscale wire. The “determination” may be quantitative and/or qualitative, depending on the application. The term “reaction entity” refers to any entity that can interact with an analyte in such a manner to cause a detectable change in a property (such as an electrical property) of a nanoscale wire. The reaction entity may enhance the interaction between the nanowire and the analyte, or generate a new chemical species that has a higher affinity to the nanowire, or to enrich the analyte around the nanowire. The reaction entity can comprise a binding partner to which the analyte binds. The reaction entity, when a binding partner, can comprise a specific binding partner of the analyte. For example, the reaction entity may be a nucleic acid, an antibody, a sugar, a carbohydrate or a protein. Alternatively, the reaction entity may be a polymer, catalyst, or a quantum dot. A reaction entity that is a catalyst can catalyze a reaction involving the analyte, resulting in a product that causes a detectable change in the nanowire, e.g. via binding to an auxiliary binding partner of the product electrically coupled to the nanowire. Another exemplary reaction entity is a reactant that reacts with the analyte, producing a product that can cause a detectable change in the nanowire. The reaction entity can comprise a shell on the nanowire, e.g. a shell of a polymer that recognizes molecules in, e.g., a gaseous sample, causing a change in conductivity of the polymer which, in turn, causes a detectable change in the nanowire. The term “binding partner” refers to a molecule that can undergo binding with a particular analyte, or “binding partner” thereof, and includes specific, semi-specific, and nonspecific binding partners as known to those of ordinary skill in the art. The term “specifically binds,” when referring to a binding partner (e.g., protein, nucleic acid, antibody, etc.), refers to a reaction that is determinative of the presence and/or identity of one or other member of the binding pair in a mixture of heterogeneous molecules (e.g., proteins and other biologies). Thus, for example, in the case of a receptor/ligand binding pair the ligand would specifically and/or preferentially select its receptor from a complex mixture of molecules, or vice versa. An enzyme would specifically bind to its substrate, a nucleic acid would specifically bind to its complement, an antibody would specifically bind to its antigen. Other examples include, nucleic acids that specifically bind (hybridize) to their complement, antibodies specifically bind to their antigen, and the like. The binding may be by one or more of a variety of mechanisms including, but not limited to ionic interactions, and/or covalent interactions, and/or hydrophobic interactions, and/or van der Waals interactions, etc.

Additionally, as discussed, a device in some aspects may include a photoresist, such as a soft photoresist. For example, in some embodiments, the photoresist may comprise a polymer formed by photo-curing a fluorinated monomer including cross -linkable function groups using a photoinitiator. This may, for example, facilitate stretchability of the device. One example of such a polymer is perfluoropoly ether dimethacrylate (PFPE-DMA). In addition, in some cases, the photoresist may be a photo-curable composition. In some embodiments, a photo-curable composition includes: a fluorinated monomer including crosslinkable functional groups; and a photoinitiator. Additional non-limiting examples of photoresist may be found in Int. Pat. Apl. Pub. No. WO 2019/084498, incorporated herein by reference in its entirety.

Some embodiments of this disclosure are directed to a photo-curable composition that can be cured to form an elastomer exhibiting high stretchability and that is chemically orthogonal to various development solvents used in photolithography and, hence, compatible with photolithography. Further, the elastomer can be patterned with fine feature resolution, and can be used as a photoresist for patterning various materials, including electrically (or electronically) active materials. Examples of applications of such photo-pattemable composition include forming stretchable and transparent substrates, stretchable and transparent dielectric/passivation/encapsulation films or layers for elastic or stretchable microelectronics, and photoresists for patterning of materials, such as in the context of implantable medical devices, wearable electronic devices, and soft electronic devices; other biomedical devices; cosmetics; prosthetics; and other applications involving an interface with a human body, an animal body, or other biological tissue where matching of mechanical properties with the biological tissue is desired.

In some embodiments, a kit may be provided, e.g., comprising a device as is discussed herein. Cells may or may not be provided with the kit. The kit may include a package or an assembly including the device, and optionally other components associated with the device, such as cells. Examples of other components include, but are not limited to, solvents, surfactants, diluents, salts, buffers, emulsifiers, chelating agents, fillers, antioxidants, binding agents, bulking agents, preservatives, drying agents, antimicrobials, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, dishes, frits, filters, rings, clamps, wraps, patches, containers, and the like, for example, for using, administering, modifying, assembling, storing, packaging, preparing, mixing, diluting, and/or preserving the device.

A kit may include instructions in any form that are provided in connection with the components of the kit in such a manner that one of ordinary skill in the art would recognize that the instructions are to be associated with those components. For instance, the instructions may include instructions for the use, modification, mixing, diluting, preserving, administering, assembly, storage, packaging, and/or preparation of the device. The instructions may be provided in any form recognizable by one of ordinary skill in the art as a suitable vehicle for containing such instructions, for example, written or published, verbal, audible (e.g., telephonic), digital, optical, visual (e.g., videotape, DVD, etc.) or electronic communications (including Internet or web-based communications), provided in any manner.

The following documents are incorporated herein by reference in their entireties: U.S. Provisional Patent Application Serial No. 62/865,648, filed June 24, 2019, entitled “Organoids Containing Electronics, and Methods Thereof,” by Liu, et al. U.S. Provisional Patent Application Serial No. 62/872,031, filed July 9, 2019, entitled “Organoids Containing Electronics, and Methods Thereof,” by Liu, et al. ; U.S. Patent No. 7,211,464, issued May 1, 2007, entitled “Doped Elongated Semiconductors, Growing Such Semiconductors, Devices Including Such Semiconductors, and Fabricating Such Devices”; U.S. Patent No. 7,301,199, issued November 27, 2007, entitled “Nanoscale Wires and Related Devices”; and International Patent Application No. PCT/US2010/050199, filed September 24, 2010, entitled “Bent Nanowires and Related Probing of Species,” published as WO 2011/038228 on March 31, 2011. In addition, the following are each incorporated herein by reference in their entireties: U.S. Pat. Nos. 9,786,850 and 9,457,128; U.S. Pat. Apl. Pub. Nos. 2017/0069858, 2014/0073063, 2017/0072109, and 2014/0074253; and Int. Pat. Apl. Pub. No. WO 2019/084498.

Also incorporated herein by reference in their entireties are U.S. Pat. Apl. Pub. No. 2022-0213425, Int. Pat. Apl. Pub. No. WO 2020/263772, U.S. Pat. Apl. Ser. No. 63/567,834, and U.S. Pat. Apl. Ser. No. 63/681,224.

The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

EXAMPLE 1

Soft and stretchable bioelectronics for minimally invasive brain implantation via embryonic neurulation. During vertebrate development, the neural plate, a 2D single-cell ectoderm-derived layer on the surface of the embryo, folds to form the neural tube, and, with further expansion and more folding, morphs into the 3D brain and other portions of the nervous system. It is believed that this 2D-to-3D reconfiguration process could be leveraged to distribute appropriately designed soft, stretchable bioelectronics throughout the brain with minimal impact on brain development or function. It was initially sought to realize this hypothesis in the Xenopus leaves (frog) embryo (FIG. 4A), given that it is a widely used in developmental biology and because its developmental processes are well understood. Moreover, the neural plate is exposed to aqueous solution during Xenopus embryogenesis, making it accessible for the placement of a bioelectronic device prior to the neural tube formation. (Xenopus embryos and tadpoles which have embedded microelectronics will be referred to herein as cyborg frog embryos and cyborg frog tadpoles, respectively. Control tadpoles and embryos contain no embedded microelectronics.) FIG. 4B and 4C show two schematic views of how neural development drives the integration of an appropriately designed device. Mesh microelectronics containing a stretchable electrode array is implanted non-invasively driven by the 2D-to-3D reconfiguration of neural tissue during neurulation (FIG. 4B). While forming a 3D fully integrated structure with neural networks throughout the entire brain (FIG. 4C), the stretchable electrode array reveals the brain- wide electrophysiological evolution over the course of development of Xenopus leaves (FIG. 4A).

It was aimed to restrict device implantation to only the cranial neural plate, as this region ultimately forms the brain (the caudal neural plate extends axially to form the spinal cord). Based on calculations, it is believed that the formation of the frog embryo brain should introduce less than 30% axial strain to the device over the course of development (see Methods). Therefore, the first design featured 40-nm-thick chromium/gold serpentine interconnects and an 800-nm-thick SU-8 encapsulation layer, an approach that can tolerate up to 30% strain in developing organoids in vitro. However, the elastic modulus of frog embryos at the neurulation stage is significantly lower than that of organoids (FIG. 11A); the SU-8 device, was too stiff, cutting through the neural plate shortly after integration and critically damaging the embryo (FIG. 1 IB, FIG. 11C). The bending stiffness of each device layer can be calculated as proportional to Ebh, where E, b, and h are the elastic modulus, width, and layer thickness, respectively. The SU-8 encapsulation layer dominated overall device flexibility. This was in large part due to the SU-8 layers’ thicknesses and SU-8’s intrinsic stiffness.

It was therefore hypothesized that a stretchable device with softer dielectric layers would have greater overall flexibility and therefore be less likely to damage the embryonic tissue. A 1-pm-thick mesh structure made of a softer elastomer was designed and tested: styrene-ethylene-butylene-styrene (SEBS). This mesh was successfully embedded into the neural tube during neurulation without obvious damage to the embryo (FIG. 1 ID). SEBS, however, is incompatible with sub-micrometer multilayer photolithography in some cases, which can limit the fabrication of stretchable mesh electronics. Therefore, a perfluoropolyether-Di methacrylate (PFPE-DMA) -based photoresist was developed, which possesses both low elastic modulus and intrinsic stretchability (FIG. HE, FIG. 11F) as well as the chemical orthogonality needed for nanofabrication. By tuning the molecular weight of the PFPE-DMA precursor, the elastic modulus of the photopatterned PFPE-DMA film (FIG. 4D) could be altered. More flexible PFPE-DMA films better matched the mechanical properties of brain tissue but were also more difficult to handle during fabrication and implantation. PFPE-DMA was ultimately chosen with a molecular weight of 8 Kad and an elastic modulus of ~0.3 MPa (FIG. HE). For comparison, the elastic modulus of the stage 15 embryo and brain tissue are 74.10 +/- 2.45 Pa and -1 kPa, respectively (mean +/- s.e.m., n = 100, FIG. 11A); and the elastic modulus of SU-8 and SEBS are -4 GPa and -17.65 MPa, respectively. In hysteresis testing, 8 kDa PFPE-DMA film maintained stable linear elasticity through 50 cycles of 50% uniaxial stretching (FIG. 4F). Finally, 8 kDa PFPE-DMA- encapsulated gold serpentine ribbons maintained their structure under 50% uniaxial stretching (FIG. 4G).

To further evaluate the potential impact of device stiffness on embryonic brain implantation, PFPE-DMA and SU-8 encapsulated serpentine ribbons using finite element analysis (FEA) were compared. FEA showed that when stretching 1-pm-thick PFPE-DMA and SU-8 ribbons encapsulating 40-nm-thick gold to a 29% strain, close to the relative extension of the Xenopus brain during early- stage development, the maximum stress in PFPE-DMA was only 68 kPa while the maximum stress in SU-8 ribbon reached 759 MPa (FIG. 4H). As the yield strength of PFPE-DMA is 0.45 MPa (>> 68 kPa, FIG. 1 IE), and the yield strength of SU-8 is 80 MPa²⁵ (<< 759 MPa), the PFPE-DMA ribbon remained in the elastic deformation regime, while the SU-8 ribbon would likely fail. When the two ribbons were stretched to the same maximum stress of 100 kPa, close to the limit that the embryonic tissue can sustain, the PFPE-DMA ribbon reached a 39.48% strain (FIG. 41), exceeding embryonic axial deformation during development. In contrast, the SU-8 ribbon barely stretched at all (0.00000031% strain), indicating that it is too stiff to deform alongside with the embryo. In addition, FEA simulation of the interaction between each mesh and the embryonic neural plate in the neurulation (FIG. 4J, FIG. 4K and FIGs. 12A-12B, see Methods) showed that the 1-pm-thick PFPE-DMA mesh encapsulating 40-nm-thick gold introduced a maximum force of 0.1 pN and maximum stress of 10 Pa to the neural plate. The SU-8 mesh introduced a maximum 1 mN of force and 100 Pa of stress. In total, therefore, FEA suggested that any mechanical deformation or damage to the neural plate during implantation resulting from PFPE-DMA meshes would likely be much lower than those caused by SU-8 meshes.

In addition to the mechanical compatibility, PFPE-DMA shows superior contact properties with embryos in comparison to SU-8. When implanted, SU-8 devices adhered to embryo cells, scraping cells from the embryos during implantation (FIG. 11B). In contrast, PFPE-DMA devices displayed a lower surface free energy (the surface free energy of PFPE- DMA is 21.2 mJ/m², while that of SU-8 is 29.5 mJ/m², FIG. 11G) and therefore stick less to cells (contact angle of cytomembrane analog with PFPE-DMA is 23.66° +/- 1.24°, while the contact angle of cytomembrane analog with SU-8 is 0°, mean +/- s.e.m., n = 5, FIG. 11H). Experimental testing supported previous measurements and simulations: a 1-pm-thick PFPE- DMA stretchable mesh can be successfully implanted into the embryonic brain with no obvious damage (FIG. 111).

Device structures that would minimize impact on embryonic development when integrated via this method were tested (FIG. 5A). First, to accommodate the neural plate’s folding, the device needed to maintain close contact with neural plate cells. This can help ensure that the device embeds into the neural tube as development progresses. The interconnects of the device were deformable to accommodate the 3D re-organization of the neural tube and eventually to allow device integration into the 3D structure of the tadpole brain. It was therefore concluded that the device should contain: (i) polymeric thin-film blockers to keep the stretchable mesh electrodes away from the caudal neural plate (elongates to form the spinal cord) (FIG. 5A and FIG. 11 J, FIG. UK); (ii) stretchable ribbons anchored on the substrate to hold the electrodes at the correct portions of the neural plate (FIG. 5A and FIGs. 11K-11L); and (iii) face-down electrodes connected via fully encapsulated stretchable interconnects (FIG. 22A and FIG. 33A).

Nanofabrication of soft, stretchable mesh microelectronics. It was sought to implement PFPE-DMA as the encapsulation layer in the fabrication of a functional stretchable mesh device. A typical microfabrication protocol was adapted to allow the photopatteming of multilayered ultra-thin PFPE-DMA structures as follows (FIG. 5B, FIG. 5C, see Methods): (i) As dimethacrylate polymerization is sensitive to oxygen, the conventional mask aligner was contained in a nitrogen chamber to prevent spin-coated PFPE- DMA films from oxygen exposure (FIGS. 13A-13D); (ii) Photoresist and metal were initially difficult to deposit on PFPE-DMA films (FIG. 13E) due to its low surface energy (FIG. 11G). To improve surface adhesion, the PFPE-DMA surface was treated with argon gas plasma, which allowed for photopatteming of standard photoresist and metal deposition on the PFPE-DMA layer to form interconnects (FIG. 13F , FIG. 22B, and FIG. 33B); (iii) The platinum (Pt) electrode array was patterned as the bottom layer (FIGs. 5B, 5C and FIG. 13G) to allow direct contact with neurons during implantation.

The mechanical properties of the PFPE-DMA device were evaluated. Photographic and bright-field (BF) microscopic images of the device (FIGS. 5D-5H) showed that the PFPE-DMA passivation and gold interconnects were successfully patterned with micrometer resolution. Scanning electron microscope (SEM) images (FIG. 14A, FIG. 14B) and atomic force microscope (AFM) topographic images (FIG. 14C, FIG. 14D) showed smooth PFPE- DMA passivation surfaces without discernible cracks or flaws. SEM images of the device cross-section (FIGS. 5I-5L) showed that the gold interconnects were fully encapsulated by the PFPE-DMA passivation. SEM, AFM, and BF microscopic images indicated that the PFPE-DMA encapsulated gold interconnects self- wrinkled at multiple scales: anisotropic global wrinkles at the micrometer scale (FIG. 5J) perpendicular to the longitudinal direction of gold interconnects (FIG. 14E) and isotropic local wrinkles at the sub-micrometer scale (FIG. 5L and FIG. 14F, FIG. 14G). Together, the multi-scale wrinkles give the gold interconnects with a high degree of stretchability. After fabrication, the device was released from the substrate (FIG. 13H). Free-floating stretchable mesh device showed no clear damage when stretched, bent, or twisted (FIG. 5M). After stretching and bending, SEM images revealed the device ribbons remained intact with passivation (FIG. 14H-14J). Impedance testing demonstrated that the fully encapsulated interconnects sustained conductivity after bending, 33% longitudinal or 38% transverse uniaxial strain (FIG. 6A and FIG. 14K, 14L). In three-point bend testing using AFM, the device introduced negligible additional force when embedded inside a 100- m-thick gelatin membrane as compared to gelatin with no embedded device (FIG. 6B, see Methods), suggesting tissue-level softness and stretchability.

The electrical performance and in vitro biocompatibility of stretchable electronics was characterized. Electrodes were electroplated with Pt black to further reduce their electrochemical impedance for in vivo recording (FIG. 14M). Electrode impedance was consistent from batch-to-batch (FIG. 14N), indicating the robustness of sensor fabrication procedures. Continuous measurements of device electrochemical performance showed stable impedance over the 12-day time course of in vitro incubation (FIG. 6C) and over the 7-day time course of in vivo implantation (FIG. 6D). A 10-day in vitro co-culture of the PFPE- DMA or its potential degradation products with wild-type rat cortical neurons did not result in significant change in the live/dead cell ratio of the neurons (n = 6, p > 0.05; FIG. 140. All statistical tests performed were two-tailed, unpaired, t-tests unless specified otherwise; actual p values are reported in FIG. 30 and FIGS. 41A-41B). Together, the longevity, stretchability and in vitro biocompatibility of the mesh microelectronics were sufficient to move forward with implantation and in vivo electrophysiological interrogation throughout Xenopus development.

Brain-wide, non-invasive implantation of stretchable mesh electronics via embryo development. Next, the PFPE-DMA-based mesh electronics were implanted into the frog embryo at the early stage of neurulation, Nieuwkoop and Faber (NF) stage 15. Time-lapse bright-field imaging (FIG. 7A) depicted the gradual internalization of the stretchable mesh electronics by the neural plate as the neural plate transforms into the neural tube during stages 15 to 19. Images from stages 30 to 47 (FIGs. 7B-7D) showed that when the embryo developed into a tadpole, the stretchable meshes were completely embedded into the brain without interrupting its development. A flexible cable connected the electrode array to the exterior of the brain for data collection. Once the devices were released from their substrates, cyborg frog tadpoles could swim freely with the embedded device. Cyborg frog tadpoles exhibited normal development up to later stages, with no significant differences in morphological features (FIG. 34A), survival rates (FIGs. 34B), or developmental timing (FIG. 34C) compared to the control group. These results indicate that the implantation of soft bioelectronics does not interfere with embryonic development, maintaining both the viability and typical growth patterns of the tadpoles. Thus, our bioelectronic system is confirmed to be biocompatible and safe for use in developmental studies.. To characterize the 3D distribution of mesh electronics within the tadpole brain, frog tadpoles were fixed, cleared, whole-mount-stained, and imaged (details of antibody usage in this and subsequent staining experiments are provided in FIG. 31). Whole-mount-staining maximizes the chance that the brain-microelectronics system would remain intact throughout imaging (FIG. 15A). The tadpole was fixed (FIG. 7E), cleared the tissue by removing the pigment and lipids, and stained cell nuclei with 4',6-diamidino-2-phenylindole (DAPI), neurons with HuC/D, and electronics with Rhodamine 6G (R6G). 3D reconstructed confocal fluorescence microscopic imaging (FIG. 7F) showed that the mesh electronics embedded across multiple brain regions. Zoom-in images (FIG. 7G, FIG. 7H, FIG. 34C) further illustrated the integration of the mesh within the 3D structure of the neural tissue. Th e device appears embedded into the forebrain, midbrain, and hindbrain, forming close contact with neurons (FIG. 15C and FIG. 24A). When the device was implanted at a later stage of neurulation (stage 16), the device did not appear in the neural tube but only superior to it (FIG. 15D), demonstrating the necessity of integrating the mesh with the neural plate at the beginning of neurulation. Cell-type- specific protein marker staining was carried out, imaging transverse plane cryosections of the fore-, mid-, and hindbrains of tadpoles fixed at 2-, 4-, and 8- days post fertilization (DPF) (FIG. 15B). The basal body, neurons, and neural stem cells were stained with acetylated-tubulin, myelin transcription factor 1 (Mytl), and SRY-box transcription factor 2 (Sox2), respectively. Confocal fluorescence microscopic images further confirmed device integration into brain tissue (FIG. 15E and FIG. 24B).

To assess the impact of the embedded devices on cell proliferation and differentiation, and any potential immune response during development, the cell number (FIG. 25 A) and fluorescence intensity (FIG. 25B) of cyborg frog tadpoles were compared with control frog tadpoles from cryosection fluorescence images at multiple time points during development. Results showed no statistically significant difference in neural stem cell or neuron numbers between cyborg and control tadpoles (for each brain region of a specific day, n > 3 tadpoles, p > 0.05;, FIG. 71 and FIG. 7 J). Additionally, inflammation markers were selected based on the following criteria: (i) their use in well-established studies related to Xenopus laevis, and (ii) their documentation in the Xenopus laevis gene expression database²⁸. The fluorescent intensity of the selected markers — Aldehyde dehydrogenase 1 family member 1 (Aldhlllf and vimentin²⁹, which quantify astrocyte numbers, and bromodeoxyuridine (BrdU)³⁰, which quantifies cell proliferation at lesion sites — showed no statistically significant difference between control and cyborg tadpoles (n > 6 tadpoles, p > 0.05; FIG. 7K-7M, see Methods) .In addition, aldehyde dehydrogenase 1 family member 1 (Aldhlll). vimentin and bromodeoxyuridine (BrdU) staining that quantify astrocyte numbers and cell proliferation at the site of a lesion, showed no statistically significant difference between control and cyborg tadpoles, demonstrating an immune response-free implantation (for each brain region, n > 6 tadpoles, p > 0.05; FIG. 7K, FIG. 7M, see Methods).

To further investigate the effects of the embedded electronics on embryo development, quantitative polymerase chain reaction (qPCR) was performed on cyborg and control embryos at 8-DPF. Stress genes which have previously been documented in Xenopus laevis gene expression profiles were targeted. These findings revealed no significant differences between control and cyborg embryos in the expression levels of stress genes among the genes that were assessed (FIG. 7N), supporting the notion that the presence of embedded microelectronics had a minimal impact on the developmental processes of the embryos.

To further assess device integration impact on Xenopus development, frog tadpole behavior was assessed using three well-established behavioral tests (FIG. 16A-16I, see Methods): (i) color preference to characterize the visual function based on the preference of frog tadpoles to stay on the white side of a bicolored tank; (ii) visual avoidance to characterize the maturation of visual responses in the optic tectum based on the ability of tadpoles to avoid incoming obstacles; and (iii) edge preference to test the motor behavior of tadpoles based on their preference and ability to move along the edge of a container. Analyses of behavior data showed that at 9-DPF, cyborg and control tadpoles (i) had no statistically significant difference in their preferences to stay in the white half of a bicolored tank (control vs. cyborg tadpoles, 92.8% vs. 91.2%, n > 4 tadpoles, p > 0.05, FIG. 16J); (ii) both avoided black dots introduced by a screen with no statistically significant difference in their success ratio (control vs. cyborg tadpoles, 100% vs. 97.2%, n > 4 tadpoles, p > 0.05, FIG. 16K); and (iii) swam along the edge of their container (the outmost quarter of the radius) at the same speed (~0.4 mm/s) with no statistically significant difference in the proportion of time spent at the edge (control vs. cyborg tadpoles, 93.2% vs. 93.9%, n > 4 tadpoles, /? > 0.05, FIG. 16L).

These behaviors were further assessed in control and cyborg tadpoles at 1- to 9-DPF on a daily basis. Data from 1- to 3-DPF were not included, as tadpoles still could not swim. Results showed no statistically significant difference between cyborg and control tadpoles at any stage of development (for each day, n = 6 tadpoles, /? > 0.05; FIGs. 7O-7Q). Notably, both cyborg and control tadpoles began to display the color preference at 7-DPF (from 6- to 7-DPF, n = 12 tadpoles, p < 0.0001; FIG. FIG. 70), visual avoidance at 7-DPF (from 6- to 7- DPF, n = 12 tadpoles, p < 0.0001; FIG. FIG. 7P), and edge preference at 6-DPF (from 5- to 6- DPF, n > 8, p < 0.0001; from 6- to 7-DPF, n = 12, p < 0.01; FIG. 7Q). In total, behavioral assessment suggested that the implanted devices did not introduce a significant perturbation to the development of the tadpole visual and motor systems, nor did they impact tadpole visual and motor system function.

Continuous tracking of neural electrophysiology over the course of brain development. Subsequently, whether the integrated electronics allowed continuous electrical recording from the developing brain, and benchmarked signals with previous terminal measurements at various developmental stages, were tested. Cyborg tadpoles were cultured in an oxygen anesthetic system as previously reported to minimize the tadpole movement for recording (FIG. 17A). At this point, it was difficult to record from behaving tadpoles due to the restriction of tadpole motion by the flexible cable which connects the embedded electronics to the data acquisition system. The oxygen anesthetic system that was adapted has been shown not to interrupt tadpole development. The mesh electronics were connected via interconnects and VO pads to the amplification and data acquisition system using a flexible cable. Electrical recording was conducted in a Faraday cage (FIG. 17B).

Electrical signals from the developing embryonic brains were continuously recorded. Recordings from the same embryo at different timepoints showed the evolution of stagespecific electrical activities at different development stages (FIGs. 8A-8O and FIG. 17C). In a representative experiment, at stage 24, spontaneous slow oscillations were recorded (amplitude: 941.0 +/- 73.4 pV, width: 1.44 +/- 0.07 s, interval: 3.26 +/- 0.10 s, mean +/- s.e.m., n = 3 tadpoles) from four electrodes distributed across different brain regions (FIGs. 8A-8C). Analysis of the activation time delay across the channels (FIG. 8D, FIG. 8E, see Methods) showed that the oscillation waves maintained a constant propagation direction, from the forebrain to the midbrain, suggesting that brain- wide electrical activities were synchronized at this stage. Then, at stage 26, faster calcium wave-like signals emerged (FIGs. 8F-8H and FIG. 18A, FIG. 18B, amplitude: 163.7 +/- 5.2 pV, width: 132.3 +/- 3.0 ms, interval: 1.9 +/- 0.1 s, mean +/- s.e.m., n = 3 tadpoles). The width and interval of the waveforms were consistent with recordings of spontaneous localized calcium release from cells reported previously (FIG. 17F, FIG. 17G). Cross-channel examination of the temporal dynamics of these fast waves across different channels did not indicate a stable time delay, potentially indicating the gradual increase of localized activities in the brain (FIG 81, FIG. 8J, see Methods). Finally, at stage 40, both local field potential (LFP)-like signals and fast spikes were observed in the brain (FIGs. 8K-8M). By applying a 300-3,000 Hz bandpass filter, single-unit action potential-like fast spikes were isolated using a conventional spike sorting algorithm (FIG. 8N, see Methods). The widths of the action potential (amplitude: 31.00 +/- 0.69 pV, width: 2.04 +/- 0.04 ms, interval: 41.83 +/- 3.15 ms, mean +/- s.e.m., n = 3 tadpoles) were comparable with previously reported data (FIG. 17H). Drug testing ([2R]-amino-5- phosphonopentanoate [APV], cyanquixaline [CNQX], bicuculline [BIC], picrotoxin [PTX], and Tetrodotoxin [TTX]) confirmed that these spikes came from the electrical activities of neurons (FIGs. 18C-18G). After recording, the tadpoles were fixed, cryosectioned, and stained to identify electrode position in the brain (FIGs. 18H-18O). Images showed that electrode which recorded single-unit action potential-like signals formed direct contacts with neurons in the brain (FIGs. 18L-18O).

Continuous electrophysiology over the course of axolotl brain development with high- density electrode array. Next, the number of channels was expanded in the mesh electronics and assessed their compatibility with various animal models. To accomplish these objectives, a high-density electrode array was integrated into mesh electronics and co-developed it into the brain of the Amby stoma mexicnum (axolotl) model, as axolotl is a unique model reflecting both neurulation development and regeneration. The electrode array was configured as tetrodes, with interelectrode distances set as 50 micrometers (FIG. 26), comparable to the size of a single neuron soma, to ensure that each neuron can be simultaneously recorded by multiple nearby electrodes, improving spike sorting accuracy and enabling tracking of the neurons in developing tissue.

To fabricate the high-density electrode array, electron beam lithography was employed to decrease the size of the gold ribbon to the nanoscale level (FIG. 19A). The final design featured a face-down 32-channel electrode array with 500 nm gold interconnects width (FIG. 9A). BF microscopic images of the electrode array (FIGs 9A-9E) showed that the PFPE-DMA passivation and gold interconnects were successfully patterned with nanometer resolution. Mechanical tests after device releasing showed the free-floating stretchable electrode array remained intact even when subjected to stretching, bending, or twisting (FIG. 19B).

The high-density mesh electronics were continued to be implanted into axolotl embryos at the equivalent stage as had been done in frog embryos (normal stage 15). Timelapse BF imaging (FIG. 9F) illustrated the gradual internalization of the stretchable mesh electronics as the neural plate formed into the neural tube (stages 15 to 24). Subsequent images (stages 29 and 33, FIG. 9G, FIG. 9H) revealed that as the cyborg axolotl embryo developed into a tadpole, the stretchable meshes became fully embedded within the brain. Subsequently, the electrophysiology recording process was initiated. Cyborg axolotl embryos and tadpoles were continuously monitored in culture media, with their slight movements accommodated by the stretchable interconnects, maintaining recordings until stage 38. During the continuous recording of a representative axolotl embryo, spike signals emerged at stage 27 (FIG. 91- 9L).

Over the following five days (corresponding to stage 27, 28, 29, 31, 33 respectively), a total of 478,715 spikes (FIG. 9N) were recorded from 20 well-isolated individual neurons (silhouette score45 = 0.6047), distributed across various electrodes (FIG. 90), whose clusters were visualized by uniform manifold approximation and projection (UMAP) (FIG. 9P). The average spike waveforms recorded by each electrode were overlaid with the electrode array layout, confirming that spikes from each neuron were consistently recorded by multiple nearby electrodes, with amplitude variation attributed to neuron-electrode distances (FIG. 21A). By mapping electrode position and respective waveform amplitudes, the relative position of neurons to the electrode array was estimated. This approach allowed for the observation of neuron location changes within the high-density electrode array during brain development (FIG. 19G-19I).

Exploring brain processing of distant injury using high-density electrode arrays Following the continuous recording, a spinal cord injury was induced in the cyborg axolotl embryos. Notably, post- injury, the axolotl brain exhibited a significant increase in neuronal firing rate, resembling the neural activity observed during early brain developmental stages when neurons first begin firing (FIG. 21B). This suggested that brain activity might play a role in spinal cord regeneration and motivated further investigation into the brain processing of distant injuries.

Leveraging the electrophysiology tool’s ability to track neural population activity at singleunit, single-spike resolution, the brain neural dynamics of axolotl embryos undergoing distant injury — tail amputation and regeneration were assessed. In this experiment (FIG. 10A), 64- channel electrode arrays were implanted in axolotl embryos during neurulation allowing for seamless integration with 3D brain tissue. The embryos were divided into test and sham groups with the test group undergoing tail amputation at stage 30 and the sham embryos left uninjured. A representative continuous recording identified 31 well-isolated units distributed across electrodes (FIG. 10B) and spanning the recording period (FIG. IOC), whose clusters were visualized by UMAP (FIG. 21C). Analysis of unit firing rates (FIG. 10D) showed significant increases in firing rate post- amputation (Time 36, 48, 60 and 72 hours; average firing rates of 13.09, 18.59, 16.07, and 10.23 spikes/s, respectively) compared to pre- amputation rates (Time 0, 12 and 24 hours; average firing rates of 0.17, 0.64, and 0.14 spikes/s, respectively) (FIG. 21D). In contrast, sham embryos displayed stable firing rates across the same developmental stages. Normalized unit firing rates (FIG. 21E) revealed a significant increase in unit presence immediately following tail amputation.

Tail amputation also alters neural population dynamics. Pearson correlation coefficients among unit firing rates (FIG. 10E) revealed that post-amputation units (average correlation coefficient is 0.1785 ± 0.0526, mean ± s.d., n = 930) exhibited higher correlated activity compared to pre- amputation units (average correlation coefficient is 0.0100 ± 0.0520, mean ± s.d., n = 930). Additionally, factor analysis of unit firing rates was used to characterize neural states (FIG. 10F), revealing a significant drift in latent space after tail amputation. This drift first progressed further and then partially reverted.

To test whether these neural activity changes contribute to regeneration, external electrical stimulation was applied to the axolotl brain via brain-embedded electrodes after tail amputation. To achieve effective neuromodulation, all the electrodes were utilized during stimulation. External electrical stimulation was delivered as pulses (0.9 V, 10 Hz, 2 ms square wave), mimicking neural activity patterns characteristic of the post-amputation stage. Axolotl embryos implanted with mesh electronics underwent tail amputation at stage 30. Test cyborg embryos received continuous electrical stimulation (FIG. 10G), while the control did not. Analysis of regenerated tail length (FIG. 10H) showed significantly higher regeneration rates in the stimulated group compared to the sham group (from days 1 to 3 post-amputation, tail length in sham vs. stimulated groups: day 1, 0.082 ± 0.029 vs. 0.14 ± 0.028; day 2, 0.13 ± 0.044 vs. 0.31 ± 0.047; day 3, 0.28 ± 0.056 vs. 0.37 ± 0.042; day 4, 0.35 ± 0.042 vs. 0.41 ± 0.088 mm; mean ± s.d., n = 5). By day 4, the difference diminished as tail regeneration neared completion. The experiment demonstrates that external electrical stimulation in the brain region can accelerate regeneration in the tail region. To ensure reliable isolation of individual sorted units, the interspike interval (ISI) variability and autocorrelograms of spikes were further examined, both of which confirmed successful isolation of distinct units (FIG. 20A). Furthermore, the average spike waveforms from multiple recording days were overlapped, captured simultaneously by multiple electrodes, with the layout of the corresponding electrode array. This visualization shows that spikes originating from the same neurons could be consistently recorded by multiple nearby electrodes, exhibiting varying amplitudes over time and space (FIG. 9M and FIG. 20B). Leveraging the spatial distribution of the electrodes and respective mean waveform amplitudes at each electrode, the relative position of neurons relative to the electrode array was estimated. As a result, 20 well-isolated individual neurons could be discerned (silhouette score = 0.6047), their single-unit spikes monitored, and any changes in their positions observed over the recording days using the tetrode-like mesh electronics (FIGs. 19G-19I). Collectively, these results demonstrated the ability of the mesh electronics with a high-density electrode array to effectively track singleunit action potentials from the same neurons over an extended period.

A novel material, structural designs, and fabrication methods were developed for tissue-level-soft, stretchable, and embryo-development-compatible electronics. A new passivation material, PFPE-DMA, compatible with both photolithography and e-beam lithography, was implemented to match the softness of embryos as well as to create high- density electrode arrays suitable for brain integration during neurulation and long-term tracking of single-unit action potentials. These advances allow high-yield integration (FIG. 42) of the electrode array into the developing brain by leveraging the 2D-to-3D reconfiguration of its nervous system during embryonic development This example developed novel material, structural, and fabrication methods for the design of tissue-level- soft, stretchable and embryo-development-compatible electronics. These advances allow the high yield integration (FIG. 32) of an electrode array into the developing brain by leveraging the 2D-to-3D reconfiguration of its nervous system during embryonic development. Immunostaining, fluorescence imaging, gene expression analysis, and behavioral testing yielded no discernable perturbations to brain development or function. The implanted device allowed long-term stable tracking of brain electrophysiology at cellular and millisecond spatiotemporal resolution from the same embryo throughout organogenesis. This work constitutes an entirely novel method for the implantation of soft electronics into a living organism throughout the 3D organ.

The device was validated by continuously recording from the developing embryonic brain in two vertebrate species: frog and axolotl embryos. Frog embryonic recordings showed evolving brain- wide electrical activities during development (FIG. 80 and FIG. 18P, FIG. 18Q): From stage 20 (FIGs. 18R-18T) to 24, slow-wave synchronized electrical activities propagated across the neural tube from the forebrain to the midbrain. These synchronized signals gradually decouple as calcium wave-like signals emerged at stage 26, possibly indicating the increasing localization of brain activity. By stage 40, isolated single-unit action potential-like spikes appeared as frog tadpole brain function matured. The study in axolotl embryos indicated the developed technology holds potential not only for basic neuroscience research but also for future applications in regenerative medicine. Specifically, our brain- embedded, tissue-level stretchable electronics allowed long-term, stable tracking of cellular activities in the developing brain (FIG. 9I-9L, FIG. 9N-9P and FIGs. 198G-19I). Our results revealed a significant increase of neuronal firing rates in the brain associated with spinal cord injury in late-stage axolotl embryos (FIG. 21B). These findings suggest a potential role for the brain in participating in spinal cord regeneration and inspire further investigation into how the brain processes distant injuries and potentially directs their repair. To explore this further, a tail amputation experiment was designed in late-stage axolotl embryos embedded with stretchable electrodes. Neural recordings before and after tail amputation revealed distinct neural dynamics in the brain’s response to distant injury. At the single-unit level, firing rates increased significantly following amputation (FIGs. 10C-10D). At the population level, neural activity exhibited higher correlations, and the overall neural state underwent substantial changes (FIGs. 10E-10F). To test whether these neural activity changes contribute to regeneration, we applied external electrical stimulation to the axolotl brain via brain- embedded electrodes after tail amputation. The results demonstrated that chronic stimulation at the elevated neural firing rate significantly accelerated tail regeneration (FIG. 10H). This finding further suggests that neural activity in the brain may play a role in directing repair and regeneration of distant tissues, and future research may compare these mechanisms to those observed in axolotl brain regeneration. Unlike traditional approaches that primarily focus on local stimulation at injury sites, the findings suggest that the brain, as a regulatory hub for distant injuries, could be targeted for therapeutic stimulation to promote systemic regeneration. These results demonstrate how the frog brain gradually develops localized neural activity during development. Axolotl embryonic recordings traced revolution of well- isolated individual neurons, and their position changes over the development. In addition, it was observed that a significant increase in neurons’ firing rate when the axolotl tadpole’s spinal cord was cut. This increase resembled the firing rate pattern observed in the early stages of development when neurons first begin to fire. The observation is consistent with the involvement of electrical activity in neuronal regeneration and the spatial transcriptomic comparison of development and regeneration. It suggests that neurogenesis in axolotls, whether during development or regeneration, may follow a similar process.

This may be scaled up for higher electrode counts. By leveraging electron beam lithography, PFPE-DMA devices were produced with a 128-channel electrode array featuring 300 nm width gold interconnect width (FIGs. 19C-19F). Further scalability can be explored through the implementation of multiplexing circuits and 3D multilayer packaging techniques. Given the similarity in neural developmental processes across vertebrates, this can be applied in other vertebrate species. In addition to frog and axolotl embryos, the incorporation of mesh electronics has been demonstrated in the developing mouse brain during embryonic neurulation (FIGs. 27A-27F, see Method). This can also be applied to rotator type in vitro embryonic culture technology, with in utero culture, or by implanting stretchable mesh microelectronics at neonatal stages. Incorporation of electrical or optical stimulators into stretchable meshes allow for the continuous modulation of neural signals during development alongside recording. The integration of sensor registration alongside other in situ characterization methods allows for the understanding how the cellular development gives rise to electrical signals. Additionally, an agarose gel fixation setup has been developed that preserves the integrity of the device VO while the tadpole swims, allowing awake recordings to be conducted (FIGs. 28A-28C and FIGs. 29A-29F, see Methods).

EXAMPLE 2

This example illustrates various methods used in Example 1.

1. Chemicals, reagents and antibodies. All chemicals were obtained from Sigma- Aldrich unless otherwise mentioned and used without further purification.

2. Brain elongation in early development of Xenopus laevis. Device axis strain from brain development was determined based on the elongation of the relevant brain regions during development. Elongation was calculated based on photos of stage 15-17 embryo neural plates and stage 45-47 tadpole brains in this work and references.

3. Preparation of the PFPE-DMA photolithography precursor. PFPE diols were obtained from Solvay and synthesized as previously reported. Then, the photo initiator ((2,4,6-trimethylbenzoyl)-phenylphosphineoxide) and bis(trifluoromethyl) benzene were mixed with PFPE-DMA to prepare the photoresist.

4. Fabrication of the PFPE-DMA device. All photoresists and developers in the nanofabrication were obtained from MicroChem Corporation. Device fabrication began with the preparation of the Ni sacrificial layer. A 3 -inch thermal oxide silicon wafer (2005, University wafer) was rinsed with acetone, isopropyl alcohol (IPA), and deionized (DI) water, and was blown dry. It was then baked at 110 °C for 3 min and treated with O2 plasma at 100 W, 40 seem O2 for 30 s. After that, hexamethyldisilazane (HMDS) was spin-coated on the wafer at 4,000 rpm/s for 1 min. Photoresist LOR 3A was then spin-coated on the wafer at 4,000 rpm/s for 1 min and hard-baked at 180 °C for 5 min. Photoresist SI 805 was spin- coated on the wafer at 4,000 rpm/s for 1 min and hard-baked at 115 °C for 1 min. Then the photoresists were exposed to with 40 mJ/cm² ultraviolet (UV) light and developed with developer CD 26 for 50 s, rinsed with DI water, and blown dry. Finally, a 100 nm Ni layer was thermally deposited on the wafer (Sharon) and lifted off in Remover PG for 3 hours.

The second step was depositing the Pt layer. A 50 nm Pt layer was deposited on the Ni sacrificial layer by electron-beam evaporator (Denton).

The third step was making SU-8 spacers. SU-8 2010 was spin-coated on the wafer at 4,000 rpm/s for 1 min and pre-baked at 60 °C for 2 min, then 95 °C for 2 min. SU-8 was exposed with 200 mJ/cm² UV light, then post-baked at 60 °C for 2 min, 95 °C for 2 min. Finally, SU-8 was developed in an SU-8 developer for 2 min, rinsed with IPA, and blown dry.

The fourth step was fabricating the bottom PFPE-DMA passivation layer. The wafer was first cleaned with acetone, IPA, water, and blown dry. Then the PFPE-DMA precursor was spin-coated on the wafer at 3,000 rpm/s for 1 min and pre-baked at 115 °C for 2 min. The PFPE-DMA was patterned with 80 mJ/cm² UV in a custom nitrogen chamber, post-baked at 115 °C for 2 min, developed in developer (bis(trifluoromethyl)benzene: 1, 1,1, 3,3- pentafluorobutane = 1:3) for 1 min and blown dry. Finally, the PFPE-DMA pattern was hard- baked at 150 °C for 50 min.

The fifth step was fabricating the gold (Au) cable layer on the top of the bottom PFPE-DMA. The PFPE-DMA surface was activated with inert gas plasma for 2-6 min. Then the photoresists, HMDS, LOR 3A, and SI 805 were patterned on the wafer as described in the preparation of the Ni sacrificial layer. After that, adhesion metal aluminum (Al) was sputtered at 250 W, 40 seem argon (Ar) for 90 s. Au was sputtered at 125 W, 40 seem Ar for 3 min (AJA International). Finally, the metal layers were lifted off in Remover PG overnight.

The last step was preparing the top PFPE-DMA passivation layer on the top of the Au cable layer. Fabrication of the top PFPE-DMA layer followed the same procedure as the fabrication of the bottom PFPE-DMA layer.

The fabrication process of PFPE-DMA with nanoscale high-density mesh electrode array was similar to the aforementioned photolithography method. The distinction lies in the fifth step where electron beam (e-beam) lithography (Elionix ELS-HS50) was utilized to pattern the Au cable on the bottom PFPE-DMA. After activating the PFPE-DMA surface with inert gas plasma for 2-6 minutes, the e-beam resist MMA EL7 was spin-coated at a rate of 4,000 rpm/s for 1 minute and subsequently hard-baked at 150°C for 90 seconds; the e- beam resist 950 polymethyl methacrylate (PMMA) A6 was spin-coated at a rate of 4,000 rpm/s for 1 minute and subsequently hard-baked at 180°C for 90 seconds. A 10 nm layer of Au was then deposited via sputtering (AJA International) to discharge during the e-beam lithography process on the PFPE-DMA. The e-beam resists were exposed to 1000 uC/cm². The Au layer was then removed using a gold etchant, and the e-beam resists were developed with Methyl isobutyl ketone (MIBK)JPA (1:3) for 1 minute. The subsequent steps involved sputtering adhesion metal Al at 250 W, 40 seem Ar for 90 seconds, and Au at 125 W, 40 seem Ar for 3 minutes (AJA International). The process concluded with the overnight lift-off of the metal layers in Remover PG.

5. Post-fabrication processing of device before implantation. Each wafer contained several devices. The wafers were first cut to isolate each device using a dicing saw. The wafer was first spin-coated with photoresist S 1813 at 4,000 rpm/s for 1 min first to protect the device during cutting. The pieces were then flood-exposed with 90 mJ/cm² UV light, developed them with CD 26 for 1 min, rinsed with DI water for 1 min, and blew them dry with N2.

The second post-fabrication step was soldering a flexible flat cable (Molex) onto the input/output pads using a flip-chip bonder (Finetech Fineplacer).

The third post-fabrication step was gluing the culture chamber onto the wafer piece. At first, a wafer piece was adhered to a microscope slide (VWR International, 48300-026) with low toxicity silicone adhesive (World Precision Instruments, KWIK-SIL) for a stable base. Then a 50 mL centrifuge tube (VWR International, 525-0610) was cut at the tail as a culture chamber. The culture chamber was glued onto the wafer piece with low toxicity silicone adhesive.

The fourth step was releasing the device. The culture chamber was filled to 5 mm with Ni etchant (TFB, Transene Company). After 5 hours, Ni etchant was washed with O.lxMMR (1 L H₂O solution contains 5.844 g NaCl (S7653), 0.1492 g KC1 (P3911), 0.1204 g MgSO₄ (M7506), 0.2940 g CaCl₂ (C1016), 1.192 g 4-(2-hydroxy ethyl)- 1- piperazineethanesulfonic acid (HEPES, H3375), 200 mg Gentamycin (VWR International, 0304), 100 mg NaOH (S8045)) for 10 times. Then the device in the chamber was incubated with 1 mL 0.01% Poly-D-lysine hydrobromide solution (P4832) overnight. After that, the device was washed with O.lxMMR three times and incubated with 1 mL 10 mg/mL O.lxMMR diluted Matrigel (Corning, 08-774-552).

6. In vitro fertilization of Xenopus laevis. In vitro fertilization of Xenopus laevis was used to get early-stage embryos for implantation. A female frog was first injected with human chorionic gonadotropin (hCG) to induce ovulation. It was placed belly-down on a table and its eyes were covered. Then, the hind legs were straightened so that the skin on the rear end was wrinkled. 500 U hCG (Merck Animal Health Company, CHORULON) was injected under the skin fold. The needle was held in the skin for 5 s before being pulled out. Then, the frog was kept at 16-18 °C. Eggs were collected 8-9 h after hCG injection once per hour. The frog was massaged to encourage ovulation into a clean 100 mm petri dish (VWR international, 470210-568). Eggs laid directly into water inhabited by the mother lose viability shortly after having been laid and should not be used. After ovulation, the frog was transferred to a clean container with system water and allowed to recover for 24 hours before being returned to the original tank.

The next step was fertilization. Before fertilization, male frogs were anesthetized with 200 g/mL benzocaine (El 501) and euthanized, and the testes were removed. Testes were stored in lx MMR at 4 °C and used within a week. Eggs needed to be fertilized right after collection. First, excess liquid was removed from eggs as much as possible. Then !4 of a testis was put into a 1.5 mL microcentrifuge tube (VWR international, 525-0990) with 1 mL of lx MMR and crushed by a plastic microcentrifuge pestle (DWK life science, 749521-1500) to release sperm. Then the sperm solution was added on top of the eggs using a transfer pipette. Eggs were mixed with sperm using pipette tips as a single layer. After 10 mins, 5 mL 1/3 x MMR was added to the mixture. Within roughly 25 minutes, the animal hemisphere of the eggs would face upward and the eggs would stick on the dish, indicating successful fertilization. Experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Harvard under animal protocol # 19-01-344-1.

7. De-jellying of Xenopus laevis embryo. The jelly around the Xenopus laevis embryos needed to be removed before implantation. The de-jelly procedure was executed right before implantation (stage 15). Embryos were put in a de-jelly solution (60 mL IxMMR contains 1.2 g L-Cysteine (168149) and 0.1 g NaOH)) and a nutator. After 5 mins, the de- jelly solution was pipetted out, and the embryos were washed with O.lx MMR for five times. After de-jellying, embryos were kept in low population density (100 embryos per 100 mL petri dish). Dead embryos were removed as soon as possible. 8. Implantation in the Xenopus laevis embryo. Before implantation, a culture chamber containing 5 mm with O.lxMMR was put under a stereoscope. A stage 15 embryo was placed inside the culture chamber. Then, the vitelline membrane of the embryo was peeled off with #5 tweezers (Fine Science Tools, 11252-40) to expose the neural plate. After that, the embryo was slid under the stretchable device using tweezers and adjusted so that the implanted ribbons overlapped with the neural plate. During sliding, one tweezer holds the device at its stretched state while the other pushes the embryo underneath.

9. Mechanical simulation. To gain more insight into the effect of the PFPE-DMA and SU-8 devices on the development of the embryo cells, finite element analysis was conducted to investigate the mechanical response of the devices under uniaxial stretch, and the devices induced stress variation in the embryo cells during the implantation and development processes. All the FEA simulations were performed using the commercial package ABAQUS 2020/Standard.

In these analyses, the PFPE-DMA and SU-8 ribbons were discretized using four-node general-purpose shell elements with reduced integration and hourglass control (S4R element type) and, the material behavior of the ribbons was captured using a linear elastic material model (with E_SU8 = 6 GPa, _s-U8 = 0.4 and E_PPPE = 500 kPa, v_PPPE = 0.4). The response of the ribbons was then simulated by conducting non-linear static simulations (*STATIC module in ABAQUS).

Although the growth of embryonic cells is a complex process impacted by many factors, the effect of the devices on the development of embryo cells was focused. Hence, a simplified growth model was employed to mimic the growth behavior of the embryo cells and investigate the stress variation in the tissue by conducting full 3D FE simulations. Specifically, the embryonic tissue was simplified and partitioned into three parts: the neural plate, ectoderm, and internal tissue, and captured the responses of the three parts using an incompressible neo-Hookean material model with strain energy density function W given by VF ^{= —} 3), where p represented the small strain shear modulus and I\ was the first invariant of the right Cauchy-Green deformation tensor. The material behavior of the embryo cell was characterized by p_plate = 150 Pa, _ectoderm = 3 kPa and p_{inside tissue} = 1-5 Pa. The cell was discretized using a non- structured mesh of 4-node linear tetrahedron elements (ABAQUS element type: C3D4H), with finer mesh size toward the neural plate where the material had undergone larger deformation. Due to the existence of buckling during the deformation, volume-proportional damping was added to the model (using the option STABILIZE in the ABAQUS STATIC module) to facilitate convergence. Specifically, the dissipated energy fraction was set to be equal to 5 X 10^-4 and the maximum ratio of stabilization to strain energy equal to 0.05. To reduce the computational cost, only half structure was simulated and applied symmetric boundary conditions to all nodes on the symmetric plane. The growth of the embryo cells was then driven by a thermal expansion of the material, relating to the thermal strain 8^th through 8^th = <z(0 — 0⁷), where a was the thermal expansion coefficient of the material, 0 was the current temperature and 0¹ was the initial temperature. In these simulations, a_piate = 0.4 [1/K], a_{inside tissue} = 0.6 [1/K], a_ectoderm = 1.0 [1/K] and the temperature gradually increased 0 until the neural plate formed a deep fold and encapsulated the device.

10. Three-point bending tests using atomic force microscopy. An atomic force microscopy was utilized (JPK nanowizard AFM) to apply three-point bending tests to gelatin membranes and gelatin membranes embedded with the PFPE-DMA mesh electronics. Samples were tested in DI water to minimize moisture loss from gelatin.

To prepare samples, 10 g/mE gelatin (G1890) was first dissolved in 60 °C DI water. The gelatin solution was then cooled to 25 °C to form a membrane, controlled by mold area and solution volume. PFPE-DMA mesh electronics were then laminated on one side of the membrane. More gelatin solution was added and cooled to embed the mesh electronics. Gelatin membranes were then glued over a 1 -mm- wide gap on 3D-printed plastic substrates. The two edges of the gap served as support points for the three-point bending test of the membrane which spanned the gap. Finally, fixed membranes were cut into several 1-mm- wide strips, some containing microelectronics and some not.

During testing, an AFM cantilever (BRUKER, SAA-SPH-1UM) was loaded onto the center of the 1x1 mm gelatin samples over the gap. The force and displacement of the cantilever were reported.

11. Elastic modulus measurement of Xenopus laevis embryonic brain tissue and organoids using atomic force microscopy. The contact mode of an atomic force microscope (AFM, JPK nanowizard AFM) was utilized to measure the elastic modulus of Xenopus embryonic tissue and organoids. Embryonic tissues were tested in O.lxMMR, and brain organoids and cardiac organoids were tested in Ixphosphate-buffered saline (PBS, VWR international, 97063-660). During the contact test, the samples were held using custom 3D- printed parts, which were specifically designed to fit the sample shape and keep them steady during the test. The AFM cantilever (BRUKER, SAA-SPH-1UM) was loaded onto the samples. The force and displacement of the cantilever were used to calculate the elastic modulus. The elastic modulus of the neural plate was considered to be the equal to that of the brain at stage 15.

12. Contact angle measurement and surface free energy measurements. The contact angles of liquid (water, diiodomethane (158429) or phospholipid (P3817)) on solid (SU-8 or PFPE-DMA) were measured by dripping a solution onto the substrate, taking a perpendicular photo, and processing the image using Fiji to determine the contact angle.

The surface free energy of solid was calculated from contact angles of water and diiodomethane by the Fowkes model: _where of and er are the dispersive and polar components of 07 (the surface free energy of the liquid), erf are the dispersive and polar components of <J_S (the surface free energy of the solid), meaning o_s = o + of ; and 0 is the contact angle of the liquid on the solid. To calculate the surface free energy of a solid o_s, of and of need to be computed individually and subsequently summed together. Diiodomethane was used to calculate o . Diiodomethane has effectively no polar component to its liquid surface tension, meaning of = 0 and o_t = o , known as 50.8 mN/m. Then of can be calculated with the measured contact angle oi dnodomethane by " = - - - . Finally, can the calculated by measuring water’s contact angle on the solid, using water’s surface free energy of = 51.0 mN/m and of = 21.80 mN/m.

13. In vitro biocompatibility test of PFPE-DMA and its potential degradation products To obtain the potential degradation products of PFPE-DMA, the PFPE-DMA mesh electronics were heated at 80 °C in a 1 M NaOH solution for 2 hours. The resulting solution, containing the products, was transferred to a 15 mF centrifuge tube and centrifuged at 500 rpm for five minutes to remove the alkaline solution. 9 mF of the supernatant was carefully removed and 9 mF of DI water added to mix with the remaining residue. The centrifugation, supernatant removal and mixing procedures were repeated for five times to obtain the final sample.

To assess the in vitro biocompatibility of the PFPE-DMA mesh electronics or their degradation products, these were mixed with the culture medium at a concentration of 1% v/v and co-cultured them with wild-type rat cortical neurons for 10 days. The live cell ratio of the neurons was then compared to that of the control group using a cell viability and cytotoxicity assay (CELL BIOLABS, CBA-240).

14. Whole-mount staining of cyborg frog tadpoles. Tadpoles needed to stay under solution and avoid contact with air during staining. Before fixing, the device was carefully cut to release the embryo. Then embryos were fixed with 4% paraformaldehyde (Thermo Fisher SCIENTIFIC, J19943-K2) overnight at 4 °C. After that, embryos were transferred to a glass dish containing a bleaching solution: (1.5 mL 30% H2O2 (H1009), 2 mL formamide (47670), 1 mL 20xSSC buffer to 35 mL DI). The dish was placed on a nutator over aluminum foil reflective backing and under fluorescent light. To remove bubbles caused by bleach, embryos were dehydrated in methanol for 5 min. Then, the embryos were rehydrated over the course of 10 minutes in stages of 80% methanol / 20% DI water; 50% methanol / 50% PBS (VWR international, 97063-660); 20% methanol / 80% PBS.

The second step was staining. Embryos were first washed with 0.1% PBST (50 mL PBS contains 50 pL Triton X-100 (X100-1L)) 2 times, 30 mins per wash. Then they were incubated in diluted CAS-Block (13.5 mL PBS containing 1.5 mL CAS-Block (Thermo Fisher SCIENTIFIC, 008120)) for 1 h at room temperature. After that, tadpoles were stained in primary antibody solution (1 mL CAS-Block containing 10 pL anti- acetylated tubulin (T7451) and 10 pL anti-HuC/D (Abeam abl84267)) for 2 days at 4 °C. Before the staining with secondary antibodies, embryos were washed with PBST for 30 min at room temperature and then blocked in PBST-CAS for 30 min at room temperature. Then the embryos were incubated in secondary antibody solution (1 mL CAS-Block containing 2 pL Alexa Fluor 488 (Invitrogen, A- 11006), 2 pL Alexa Fluor 594 (Invitrogen, A-l 1012), 20 pL Alexa Fluor 647 Phalloidin (Thermo Fisher SCIENTIFIC, A22287) and 1 pL DAPI (D9542)) for 2 days at 4 °C. Finally, the embryos were washed with PBST for 1 h at room temperature and then washed with PBS overnight at 4 °C.

15. Microscopy of cyborg frog tadpole whole-mount staining. Tadpoles were imaged in a homemade chamber. A 2 mm layer of vacuum grease (Z273554) was squeezed at the edge of a microscope slide to form the wall of a chamber. Then, a tadpole was placed in the middle of the microscope slide and merged with the mounting medium (Vector, H-1900). A cover glass was placed on the top of the vacuum grease to seal the chamber. During imaging, the tadpole was placed upside down on the confocal microscope (Leica, dMi8) and imaged with 5 pm stacks. All the images were stitched and processed with Fiji.

16. Cryosection staining of cyborg frog tadpoles. Before fixing, the device was cut carefully to release the embryo. Then embryos were fixed with 4% paraformaldehyde (Thermo Fisher SCIENTIFIC, J19943-K2) overnight at 4 °C. To perform the cryosection, tadpoles were incubated in PBST overnight at 4 °C, then in 0.1% PBST containing 15% gelatin (G1890)/15% sucrose (S7905) overnight at 40 °C. Then the tadpole was frozen in the gelatin/sucrose solution at -80 °C overnight before cryosection.

The staining protocol for anti-Sox2, anti-Mytl, and anti-acetylated tubulin was as follows: slides were placed in a wet box and incubated at 40 °C to remove residual gelatin/sucrose solution. Then, slides were washed with 0.1% PBST for 15 min at room temperature and incubated in blocking buffer (20 mL PBST containing 0.2 mL donkey serum (Jacksonimmuno, 017-000-121) and 0.8 g bovine serum albumin (Thermo Fisher SCIENTIFIC, BP1600-100)) for 1 h at room temperature. After that, slides were incubated in primary antibody solution (2 mL blocking buffer containing 20 pL anti-Sox2 (Invitrogen, 14- 9811-82), 20 pL anti-Mytl (Abeam, ab251682) and 20 pL anti-acetylated tubulin) overnight at 4 °C. Then slides were washed with 0.1% PBST three times and incubated in secondary antibody solution (4 mL blocking buffer containing 8 pL Alexa Fluor 488 (Invitrogen, A- 11006), 8 pL Alexa Fluor 594 (Invitrogen, A- 11012), 8 pL Alexa Fluor 647 (Invitrogen, A- 32787) and 4 pL DAPI) overnight at 4 °C. Finally, slides were washed with 0.1% PBST 3 times and sealed.

The staining protocol for BrdU was as follows: slides were placed in a wet box and incubated at 40 °C to remove residual gelatin/sucrose solution. Then, slides were washed with 0.1% PBST for 15 min at room temperature and incubated in blocking buffer (20 mL PBST containing 0.2 mL donkey serum and 0.8 g bovine serum albumin) for 1 h at room temperature. After that, slides were incubated in primary antibody solution (2 mL blocking buffer containing 20 pL BrdU monoclonal antibody (Invitrogen, B35128)) overnight at 4 °C. Then slides were washed with 0.1% PBST three times and incubated in secondary antibody solution (4 mL blocking buffer containing 8 pL Alexa Fluor 647 (Invitrogen, A-32787) and 4 pL DAPI) overnight at 4 °C. Finally, slides were washed with 0.1% PBST 3 times and sealed.

The staining protocol for Aidhill and vimentin was as follows: slides were placed in a wet box and incubated at 40 °C to remove residual gelatin/sucrose solution. Then, slides were washed with 0.1% PBST for 15 min at room temperature and incubated in blocking buffer (20 mL PBST containing 0.2 mL donkey serum and 0.8 g bovine serum albumin) for 1 h at room temperature. After that, slides were incubated in primary antibody solution (2 mL blocking buffer containing 20 pL anti-Aldhlll antibody (Abeam, AB56777) and 20 pL anti- vimentin (Abeam, AB16700)) overnight at 4 °C. Then slides were washed with 0.1% PBST three times and incubated in secondary antibody solution (4 mL blocking buffer containing 8 pL Alexa Fluor 594 (Invitrogen, A-11012), 8 pL Alexa Fluor 647 (Invitrogen, A-32787) and 4 pL DAPI) overnight at 4 °C. Finally, slides were washed with 0.1% PBST 3 times and sealed.

17. Microscopy of cyborg frog tadpole cryosection staining. Slides were imaged under a confocal microscope at 4,096 dpix4,096 dpi at the speed of 400 Hz. The stack thickness was 2 pm. All the images were processed with Fiji.

18. Cell counting of cyborg frog tadpole cryosection staining. Sox2 and Mytl channels were used to compare the number of neuron stem cells and neurons at different developmental stages in Xenopus laevis embryos in both the cyborg and control groups to examine the impact of implantation on development. However, directly counting the cell number from the Sox2 and Mytl images is challenging as the cells appear merged in the staining. To address the issue, DAPI was employed to identify the nuclei, allowing accurate counting of the cells.

In the first step, cells were identified with DAPI staining. To begin, local maxima were identified with prominence higher than 50 in DAPI staining images. Normally, this would lead to 4,000 to 8,000 local maxima in intensity, which covered all potential cell centers. Next, the radius of cells were estimated to be about 40 pixels manually, and local maxima that were detected within a circle of 40 pixels were suppressed to one local maximum with the highest intensity value. After filtering, each local maximum represented the cell center within at least a circle of 40 pixels in the DAPI image. In the next step, cell centers in DAPI images were overlaid on MyTl images and Sox2 images for neuron and neural stem cell counting respectively. The MyTl and Sox2 were binarized into foreground and background after Gaussian filtering. Then the DAPI images were overlaid on binarized MyTl images and binarized Sox2 images. Finally, cell centers were identified from DAPI staining in the foreground of MyTl or Sox2 staining as existing neuron cell centers or neural stem cell centers.

19. Fluorescence intensity analysis of cyborg frog tadpole immune-response images. Staining of BrdU was performed to compare the fluorescent intensity of Xenopus laevis embryos in both the cyborg and control groups to evaluate the effect of implantation on the immune response. However, because the fluorescent intensity of BrdU images is low in the control group (since no immune response is expected), and there is potential interference from the reflection of slide glass and contaminants, it was crucial to confirm that the fluorescent signals were indeed originated from the tissue. To achieve this, in the first step, the brain region was identified with DAPI staining. The DAPI images were binarized into foreground and background after Gaussian filtering. Then the binarized DAPI images were overlaid on BrdU images. The overlapping area of BrdU with binarized DAPI images were analyzed for the fluorescence intensity of BrdU.

20. Behavior tests of cyborg and control frog tadpoles. Tadpoles for behavior tests were cultured at room temperature on a white base under a 12 hours- 12 hours day-night cycle. Behavior tests were performed between 1 and 4 pm. For one trial, a tadpole was placed at the center of a clear-bottomed round tank (diameter of 12 cm) filled to 5 cm with O.lxMMR. The tank was placed on a horizontal screen and covered in a dark box. The luminance of the screen was 50 cd/m² when displaying the white color. In the test, the tadpole was stimulated by the appropriate pattern shown on the screen and its response was video recorded by a camera on top of the dark box. Any disturbance, including vibration, light, and sounds needed to be avoided during the test. Finally, the videos were processed with a custom MATLAB code to analyze the trajectory of the tadpoles.

The horizontal screen showed different patterns in color preference, visual avoidance, and edge preference. In the color preference test, the screen alternated between displaying half white and half black for 40 seconds each. Analysis of the tadpole’s trajectory revealed the percentage of time that the tadpole stayed in the white section of the tank, which was reported as the “time ratio in white side.” In the visual avoidance test, a black dot was moved towards the tadpole on the screen. Analysis of the tadpole’s trajectory revealed the percentage of successful escapes from the black dot, which was reported as the “avoidance ratio.” In the edge preference test, the tadpole swam freely on a white screen. The area within 3 cm of the tank wall was defined as the tank edge. Analysis of the tadpole’s trajectory revealed the percentage of time that the tadpole stayed in the tank edge, which was reported as the “time ratio at the side.”

21. Long-term rearing of frog tadpoles. Control and experimental groups were split evenly between 2 static 5-gallon tanks with comparable conditions. They were reared in O.lxMMR solution with partial volume water changes completed 2-3 times weekly. During water changes, evaporative loss was compensated for and the pH was buffered, with alkaline buffer (Seachem Laboratories, Inc.), as needed. Biological filtration was developed prior to animal introduction using sponge filters. Animals were fed Sera micron (Sera North America Inc.) and Fry Starter (NorthFin Fish Food). Tanks were spot siphoned between water changes. Animal health was checked daily and water quality was analyzed at least once a week to ensure it was within healthy parameters. 22. Electrode position determination. Electrode positions depicted in the “channel position” boxes in (FIG. 8E, FIG. 8J) were determined based on the mesh device design. Because the neural tube folds inwards about the spinal cord axis and not perpendicular to this axis, it was assumed that the relative axial displacement of the electrodes was maintained from device integration through embryonic development into a tadpole.

23. Electrophysiology recording and data processing. All recordings were taken with a Blackrock CerePlex Direct recording system or Intan RHD recording system. The setup was placed on an optic table and covered by a Faraday cage. For the frog embryo data, the calcium-wave like signals were analyzed with MATEAB, the spike sorting was performed using WaveClus and MountainSort. Axolotl embryo data were processed through a custom Python pipeline based on Spikeinterface.

24. Correlation analysis of continuous electrophysiology. For signals from stages 24, 26 and local field potential signals in stage 40, one group of four synchronized peaks from each channel were extracted, and Pearson correlations were calculated between each pair of the synchronized peaks. Then, Pearson correlations from all groups of synchronized peaks were pooled for each stage. Apart from local field potentials, action potentials (spikes) were also extracted for stage 40 signals. The chunks of signals from all channels were then extracted according to the time of spikes before the calculation of their pairwise Pearson correlations. East, the pooled Pearson correlations for each stage were compared.

25. Synchrony analysis of continuous electrophysiology. For signals from stages 24, 26 and local field potential signals in stage 40, the time differences between synchronized peaks in each pair of the channels were collected, generating six datasets for each stage. The standard deviation of each dataset is an indication of synchronization between two channels, with a smaller standard deviation meaning greater synchrony. The six standard deviations for each stage were then plotted and compared.

26. De-jelly of axolotl embryo. The jelly around the axolotl embryos needed to be removed before implantation. The de-jelly procedure was applied right before implantation (stage 15). Axolotl embryos (University of Kentucky, AGSC_100E) were put in lx Steinberg’s solution (1 E H2O solution contains 0.34 g NaCl, 0.005 g KC1, 0.008 g Ca(NO₃)2-4H₂O (C2786), 0.01025 g MgSO₄ (M2643), 0.056 g Tris-HCl (10812846001), 0.001 g phenol red (P3532)). Steinberg’s was prepared in lOx, stored at room temperature and used for no longer than a few months. Then, the vitelline membrane of the embryo was peeled off with #5 tweezers to expose the neural plate. 27. Implantation of axolotl embryo. Before implantation, a culture chamber containing lx Steinberg solution with Antibiotic-Antimycotic (15240062) was placed under a stereoscope. A thin layer of agar coating on the chamber can help to keep the embryo intact, it is not essential. An embryo at stage 15 was put inside the culture chamber. Then, the vitelline membrane of the embryo was peeled off with #5 tweezers to expose the neural plate. After that, the embryo was slid under the stretchable device using tweezers and adjusted so that the implanted ribbons overlapped with the neural plate. During sliding, one tweezer holds the device at its stretched state while the other pushes the embryo underneath.

28. Mouse embryo culture. To prepare culture media in advance, rat serum was collected: To collect rat serum, pathogen-free male Sprague-Dawley rats (6-8 months of age, 550-650 g) were anesthetized with isoflurane (2.5-4.0%) and maintained under anesthesia. An incision was made in the abdomen and the dorsal aorta was exposed. Then blood was collected from the aorta using a 20 g syringe, being careful to avoid hemolysis. After collection, the collecting tube was placed on ice. The rats were euthanized by thoracotomy and cutting the heart or by decapitation. Blood was then centrifuged for 20 min at l,300xg. The supernatant was collected and centrifuged for 10 min at l,300xg. The new supernatant was again collected and heat inactivated as the serum for 30 min at 56°C. The serum was filtered with a 0.45-pm filter (VWR International, 76479-020), and stored frozen at -80°C for up to 1 year.

Before embryo collection and culture, the dissection media and static embryo culture media should be prepared. To prepare the dissection medium, 45 ml DMEM/F12 (Thermo Fisher Scientific, 11320033), 4.5 ml newborn calf serum (Thermo Fisher Scientific, 26010066), and 0.5 ml Penicillin-Streptomycin (P0781), were mixed and stored in a refrigerator overnight. Prior to dissection, the medium was warmed to 37°C. To prepare the static embryo culture medium, 0.5 ml heat-inactivated rat serum, 0.485 ml DMEM/F12, 10 pl lOOx Penicillin-Streptomycin, and 5 pl HEPES buffer solution (Thermo Fisher Scientific, J16924.AE) were mixed for each embryo. The medium was sterilized using a 0.2-pm filter and stored in the refrigerator overnight. Before using the culture medium, it was equilibrated in a 37°C 5% CO2 incubator for 1 hour.

To collect the embryos, female mice (C57BL/6 mouse, timed pregnancy, 8.5 days post conception, Charles River Laboratories INC, 027C57BL/6) were euthanized, and the uterus were dissected out. Then the uterus was placed in a dish of prewarmed dissecting media and, under a stereomicroscope, removed the embryos from each decidua using forceps. Reichert’s membrane was removed from each embryo using forceps, ensuring the ectopiacental cone is left intact. The embryos were then transferred into a dish of culture media. The smallest amount of dissecting media should be transferred and the embryo should be washed if necessary. Finally, the embryos were cultured using 6-well plates in an incubator at 37°C supplied with 5% CO2 for 24 hours. Up to two embryos can be cultured per well in 1 mL culture medium, which should be covered with light mineral oil (Nidacon, NO- 100). Damaged embryos should not be used for further culture.

Experimental procedures were approved by the IACUC of Harvard under animal protocol # 19-01-348-1.

29. Implantation of mouse embryo. Prior to implantation, the culture well plate was transferred from the incubator to a 37°C heating pad. A pre- sterilized wafer piece carrying the anchored device was immersed in the media. Carefully, the embryo was placed on the wafer piece, lying on its side with the neural plate facing the device. The device was elevated using tweezers. Subsequently, the embryo was pushed toward to the device to insert the device into the neuropore. Finally, the anchor was cut, and the wafer piece was gently removed. The entire process needed to be completed within 10 minutes, and the embryos were promptly returned to culture immediately after implantation.

30. Cryosection staining of cyborg mouse embryo. Mouse embryos were taken out from culture media and soaked in 4 °C, 4% paraformaldehyde (PFA) overnight; then in 4 °C, 10% sucrose (S7905) solution overnight; and finally in 4 °C, 20% sucrose solution overnight. The soaked embryo was positioned in a cube chamber with O. C. T. compound (Tissue-Tek 4583) and frozen at -80 °C overnight. The frozen embryo was then cut into slices as needed. Before staining, microscope slides were placed in a wet box and incubated at 40 °C to remove residual O. C. T. compound. Then, slides were washed with 0.1% PBST for 15 min at room temperature and incubated in blocking buffer (20 mL PBST containing 0.2 mL donkey serum (Jacksonimmuno, 017-000-121) and 0.8 g bovine serum albumin (Thermo Eisher, BP1600- 100)) for 1 h at room temperature. After that, slides were incubated in primary antibody solution (2 mL blocking buffer containing 20 pL anti-Sox2 (Invitrogen, 14-9811-82), 20 pL anti-NeuN (abeam, AB 104224)) overnight at 4 °C. Then slides were washed with 0.1% PBST three times and incubated in secondary antibody solution (4 mL blocking buffer containing 8 pL Alexa Eluor 488 (Invitrogen, A-11006), 8 pL Alexa Eluor 647 (Invitrogen, A-32787) and 4 pL 4',6-diamidino-2-phenylindole (DAPI)) overnight at 4 °C. Linally, slides were washed with 0.1% PBST 3 times and sealed. 31. Microscopy of cyborg mouse embryo cryosection staining. Slides were imaged under a confocal microscope at 4,096 dpix4,096 dpi at the speed of 400 Hz. The stack thickness was 2 pm. All the images were processed with Fiji.

32. Agarose fixation. At first, a layer of agarose was cured on the agar scaffold of the fixation apparatus. The fixation apparatus was placed on the base with the agarose scaffold inside the agar mold. The agarose precursor was then poured into the mold, and the scaffold was taken from the mold once the agar had fully cured (FIG. 28B, state 1). Then the fixation apparatus was placed on top of the culture chamber containing the anesthetized cyborg tadpole. The design of the fixation apparatus ensured that the tadpole was positioned in the middle of the two agarose scaffolds when the apparatus was placed on top (FIG. 28B, state 2). Finally, the tadpole was fixed using a small amount of low melting point agarose, with the scaffolds serving as anchor points (FIG. 28B, state 3). The agarose used in this procedure was carefully applied to avoid covering the tadpole’s mouth or tail, allowing it to breathe and move its tail once it recover from the anesthesia (FIG. 28C). This agarose fixation must be performed at embryo stage 35 before the tadpole begins intense swimming. After fixation, the apparatus should be placed on top of the fixation apparatus to reduce media evaporation, and the media should be changed every 12 hours to increase the survival rate of the fixed tadpole.

FIGs. 4A-4K illustrate a design of soft and stretchable bioelectronics for brain implantation via embryonic development. FIG. 4A illustrates schematics showing the stepwise implantation of soft and stretchable mesh microelectronics into the brain of the Xenopus embryo via organogenesis. Mesh microelectronics track brain- wide, continuous electrophysiological evolution over the course of brain development. The mesh microelectronics with stretchable electrode array is laminated onto the neural plate at the beginning of neurulation (stage 15). During neurulation, tissue reconfiguration embeds the mesh into the neural tube (stage 24). The mesh deforms with the neural tube as it re-organizes into a 3D tadpole brain (stages 26, 40, 47). FIG. 4B illustrates schematics of zoom-in transverse sections of neural plate development showing how the mesh electronics is integrated non-invasively into the neural tube via neurulation. FIG. 4C illustrates schematics showing with further expansion and folding of the neural tube, stretchable mesh microelectronics are fully distributed and embedded throughout the 3D structure of the brain. FIG. 4D illustrates an elastic modulus of crosslinked perfluoropolyether-dimethacrylate (PFPE-DMA) with 4-, 8-, 10-, 12-kDa molecular weight. FIG. 4E illustrates an elastic modulus of stage 15 embryo, brain tissue, PFPE-DMA, styrene ethylene butylene styrene (SEBS), and SU-8. The dashed lines in the PFPE-DMA column indicate the average elastic modulus of PFPE-DMA for 4-, 8-, 10-, and 12-kDa molecular weights. FIG. 4F illustrates a stress-strain curve of 8 kDa PFPE-DMA film under 50 times cyclic, 50% uniaxial stretch loading. FIG. 4G illustrates a photographic image of 8 kDa PFPE-DMA film encapsulating serpentine gold ribbons in uniaxial stretch test. Zoom-in views show the serpentine ribbons at 0%, 10%, 20%, 30%, 40% and 50% strain state. FIG. 4H and 41 illustrate mechanical simulations revealing stress distributions in PFPE-DMA and SU-8 serpentine ribbons when they are stretched to the same strain (FIG. 4H) and the same maximum von Mises stress (FIG. 41). Dashed lines show the initial shapes of the ribbons. FIG. 4J and 4K illustrate simulations showing the force (FIG. 4J) and maximum von Mises strain (FIG. 4K) from the SU-8 and PFPE-DMA meshes applied to the neural plate during implantation and development from stages 15 to 18.

FIGs. 5A-5M and 6A-6D illustrate fabrication of tissue-level-soft stretchable mesh microelectronics for brain implantation via embryo development. FIG. 5A illustrates schematics illustrating the design of soft and stretchable mesh microelectronics for embryo implantation. The design includes a stretchable mesh electrode array for electrophysiological sensing, interconnects and input/output (VO) pads for data collection, polymeric stretchable anchors to hold the mesh to the neural plate, and blockers to restrict the mesh to the cranial neural plate. FIG. 5B illustrates schematics showing the tri-layer structure of the PFPE-DMA encapsulated stretchable mesh microelectronics: PFPE-DMA passivation layers sandwich the gold interconnects layer. The electrodes are made of platinum (Pt) and electroplated with Pt black. FIG. 5C illustrates schematics showing the fabrication steps of PFPE-DMA encapsulated stretchable mesh microelectronics. First, a nickel layer is deposited on a blank silicon oxide wafer as a sacrificial layer (step 1). A SU-8 layer is patterned as a spacer (step 2). Then, Pt electrodes are photolithographically patterned (step 3). Finally, the bottom PFPE- DMA (step 4), gold interconnects (step 5), and top PFPE-DMA layer (step 6) are photolithographically patterned. Zoom in figures in step 3-6 display the details of the mesh electrode arrays, highlighted in blue boxes. FIG. 5D illustrates a photographic image of representative PFPE-DMA mesh microelectronics on a glass substrate. FIG. 5E illustrates a bright-field (BF) microscopic image of the zoom-in view of the red box-highlighted region in (FIG. 5D) showing the stretchable mesh electrode array for electrophysiological recording. FIG. 5F illustrates a BF microscopic image of the zoom-in view of the red box highlighted region in (FIG. 5E) showing the stretchable interconnects. FIG. 5G illustrates a BF microscopic image of the zoom-in view of the blue box highlighted region in (FIG. 5E) showing global wrinkle structures in gold interconnects. FIG. 5H illustrates a BF microscopic image of the green box highlighted region in (FIG. 5G) showing the individual electrode. FIGs. 5I-5L, Scanning electron microscope (SEM) images showing cross-sections of PFPE- DMA encapsulated gold interconnects. Each layer is pseudo-colored and labeled. FIG. 51 illustrates a SEM image showing longitudinal cross-section along the red line in (FIG. 5H). FIG. 5J illustrates a SEM image of the zoom-in view of the red box highlighted region in (FIG. 51). A dashed line is drawn parallel to the gold layer to indicate its global wrinkles. FIG. 5K illustrates a SEM image showing transverse cross-section along the blue line in (FIG. 5H). FIG. 5L illustrates a SEM image of the zoom-in view of the red box highlighted region in (FIG. 5K). A dashed line is drawn parallel to the gold layer to indicate its local wrinkles. FIG. 5M illustrates a photographic images showing the free-floating stretchable mesh electrode array during stretching, bending, and twisting tests.

FIG. 6A illustrates resistance as a function of strain during the longitudinal stretch test of PFPE-DMA encapsulated electronics. Red dots and line plots indicate mean +/- s.d., and each gray dot and line plot represents one sample. FIG. 6B illustrates a (Left) Force as a function of displacement in atomic force microscope (AFM) three-point bending tests of gelatin membrane and gelatin membrane embedded with PFPE-DMA mesh microelectronics showing tissue-level softness and stretchability of the device. (Right) Statistics of curve slopes in the left figure. FIG. 6C illustrates an electrode impedance at 1 kHz in 37 °C PBS as a function of incubation time. Red dots and line plots indicate mean +/- s.d., and each gray dot and line plot represents one sample. FIG. 6D illustrates an electrodes’ impedance at 1 kHz as a function of post- implantation time. Red dots and line plots indicate mean +/- s.d., and each gray dot and line plot represents one sample.

FIGs. 7A-7Q illustrate a minimally invasive brain implantation of tissue-level-soft, stretchable mesh microelectronics via embryonic development. FIG. 7A illustrates time-lapse bright-field microscopic images of a representative frog embryo implanted with stretchable mesh microelectronics at different development stages showing the gradual internalization of the mesh electrode array (dashed circles) into the neural plate. FIGs. 7B-7D illustrate optical photographic images of the embryo implanted with stretchable mesh microelectronics at stages 30 (FIG. 7B), 40 (FIG. 7C), and 47 (FIG. 7D). The dashed circles highlight the interconnects outside the brain. FIGs. 7E-7F illustrate a photograph (FIG. 7E) and 3D reconstructed confocal fluorescence image (FIG. 7F) of a fixed cyborg tadpole. White dashed circles highlight the stretchable interconnects extending from the sensor array to the outside of the brain. 4',6-diamidino-2-phenylindole (DAPI) labels cell nuclei, Rhodamine 6G (R6G) labels device, HuC/D labels neurons. FIG. 7G illustrates a zoom-in image of the dashed box highlighted region in (FIG. 7F) showing the stretchable mesh microelectronics folded inside of the neural tube. FIG. 7H illustrates a zoom-in image of the dashed box highlighted region in (FIG. 7G) showing the interface between the embedded stretchable mesh microelectronics and neurons. FIGs. 7I-7M illustrate bar and dot plots showing the number of neural stem cells (FIG. 71), neurons (FIG. 7J), aldehyde dehydrogenase 1 family member 1 (Aidhill) (FIG. 7K), vimentin (FIG. 7L) and bromodeoxyuridine (BrdU) (FIG. 7M) identified from fluorescence images of the cryosection stained cyborg and control tadpoles at different developmental stages. Bar plots indicate mean +/- s.d., each dot represents one sample, two- tailed unpaired t-test, for (FIGs. 71, 7J), n = 3; for (FIG. 7K-7M), n > 6; ns, not significant. FIG. 7N illustrates a quantitative polymerase chain reaction (qPCR) targeting stress genes of cyborg and control tadpoles. Bar plots of stress gene expressions in cyborg and control tadpoles, mean +/- s.e.m., two-tailed unpaired t-test, ns, not significant. FIGS. 7O-7Q show r- t, Statistical analysis of color preference (FIG. 70, visual avoidance (FIG. 7P, and edge preference (FIG. 7Q) behavior test data from control and cyborg tadpoles at 4- to 9-days post fertilization. Bar plots indicate mean +/- s.d., each dot represents a single trial, two-tailed unpaired t-test, n > 8, **, p < 0.01, ****, p < 0.0001.

FIGS. 8A-8O illustrate continuous tracking of in vivo neural electrical activities from the same tadpole during organogenesis and brain development. FIG. 8A illustrates a schematic of the cyborg tadpole at developmental stage 24. FIG. 8B illustrates representative voltage traces from four channels in the cyborg tadpole at stage 24. FIG. 8C illustrates zoom- in views of the signals highlighted by dashed lines in (FIG. 8B). FIG. 8D illustrates a heat map of the amplitude of the signals as a function of time for the voltage traces in (FIG. 8B). FIG. 8E illustrates a spatiotemporal delay of signals across channels from the highlighted timepoints in (FIG. 8D). FIG. 8F illustrates a schematic of the cyborg tadpole at developmental stage 26. FIG. 8G illustrates representative voltage traces from four channels in the cyborg tadpole at stage 26. FIG. 8H illustrates zoom-in views of the signals highlighted by dashed lines in (FIG. 8G). FIG. 81 illustrates a heat map of the amplitude of the signals as a function of time for the voltage traces in (FIG. 8G). FIG. 8 J illustrates a spatiotemporal delay of signals across channels from the highlighted timepoints in (FIG. 81). FIG. 8K illustrates a schematic of the cyborg tadpole at developmental stage 40. FIG. 8L illustrates voltage traces from four channels in the cyborg tadpole at stage 40. FIG. 8M illustrates zoom- in views of representative single spikes highlighted by dashed lines in (FIG. 8L). FIG. 8N illustrates the average spike (mean +/- s.d.) sorted from channel 1 voltage traces in (FIG. 8L). FIG. 80 illustrates schematics illustrating how the neural activity evolves from brain- wide coordinated activity to localized neural activity and the emergence of single-unit spikes during Xenopus development.

FIGs. 9A-8P illustrate implantation and continuous recording of axolotl embryos with soft and stretchable bioelectronics with high-density mesh electrodes. FIG. 9A illustrates a schematic showing the tri-layer structure of the PFPE-DMA encapsulated stretchable mesh electronics with high-density electrode array: PFPE-DMA passivation layers sandwich a gold interconnects layer. The electrode array is constructed with 32 channels in a tetrode-like configuration. FIG. 9B illustrates a bright-field (BF) microscopic image showing the 32- channel tetrode-like electrode array for electrophysiological recording. FIG. 9C illustrates a BF microscopic image of the zoom-in view of the red box highlighted region in (FIG. 9B) showing the stretchable interconnects. FIG. 9D illustrates a BF microscopic image of the zoom-in view of the blue box highlighted region in (FIG. 9B) showing the electrode array. FIG. 9E illustrates a BF microscopic image of the zoom-in view of the green box highlighted region in (FIG. 9D) showing two individual electrodes. FIG. 9F illustrates time-lapse BF microscopic images of a representative axolotl embryo implanted with stretchable mesh electronics at different development stages showing the gradual internalization of the mesh electrode array (highlighted with dashed lines) into the neural plate. FIGs. 9G and 9H illustrate optical photographic images of the embryo implanted with stretchable mesh microelectronics at stages 29 (FIG. 9G), and 33 (FIG. 9H). The dashed circles highlight the interconnects outside the brain. FIG. 81 illustrates representative filtered voltage traces (300- 3,000 Hz bandpass filter) recorded in an axolotl. FIGs. 9J-9L illustrate zoom-in views of the voltage traces in (FIG. 91) highlighted by FIG. 9J, blue dashed lines, FIG. 9K, red dashed lines, FIG. 9L, green dashed lines. FIG. 9M illustrates representative average single-unit waveforms at each of the recording electrodes over the course of 5 days recording in cyborg axolotl tadpole. Waveforms recorded from each day are plotted as a gradient color. FIG. 9N is a raster plot of spikes sorted from continuous 5-day recording in the cyborg axolotl embryo. FIG. 90 shows the average template over the extremum electrodes of units sorted from the continuous recording in (FIG. 9N). FIG. 9P shows the uniform manifold approximation and projection (UMAP) plots across the continuous recording of sorted units in (FIG. 90).

FIGs. 10A-10H show investigating neural processing of distant injury and tail regeneration using high-density electrode arrays in late-stage axolotl embryos. FIG. 10A is a schematic illustration of the experiment designed to study neural signal changes in the axolotl brain following tail amputation and regeneration. FIG. 10B shows sorted unit (mean ± s.d.) at each of electrodes from a representative continuous recording following tail amputation and regeneration. FIG. IOC is a raster plot displaying the activity units in (FIG. 10B) over the continuous recording. FIG. 10D shows the firing rate changes of units in (FIG. 10B) throughout the recording. Gray dots and lines represent firing rate changes of individual units in the tail-amputated embryo. Red dots and lines represent the average firing rate changes of units in the tail-amputated embryo. Blue dots and lines represent average firing rate changes of units in the embryos without tail amputation. FIG. 10E shows the pearson correlation coefficient of unit spike firing rate recorded immediately before and after tail amputation. FIG. 10F shows the neural states across continuous recording derived from factor analysis of spike trains in (FIG. IOC). Each state is represented with a solid covariance ellipse, plotted on semi-transparent epoch circles. FIG. 10G is a schematic illustration of the experiment designed to investigate tail regeneration with electrical stimulation from the brain-embedded electrodes. FIG. 10H shows a comparison of tail regeneration length over time in cyborg axolotl embryo with and without electrical stimulation following tail amputation. Bar plots indicate mean ± s.d., each dot represents one animal, two-tailed unpaired t-test, n = 5, *, p < 0.05, **, p < 0.01,***, p < 0.001, ns, not significant.

FIGs. 11A-1 IK illustrate a test of implantation methods. FIG. 11A illustrates schematics showing an atomic force microscopy setup for tissue elastic modulus measurement. (Bottom) elastic modulus of stage 15, 24, 32, 40, 48 Xenopus embryos, brain organoids, and cardiac organoids. Box plots indicate minimum, lower quartile, median, upper quartile, and maximum. Each dot represents a contact measurement. FIG. 1 IB illustrates bright-field (BF) microscopic images showing broken SU-8 mesh post-implantation (left) and the embryo before (middle) and after (right) mesh implantation depicting damage to the embryo. FIG. 11C illustrates BF microscopic images showing an embryo crushed by SU-8 meshes. FIG. 11D illustrates BF microscopic images showing an embryo successfully implanted with a SEBS mesh. The dashed line circle highlights the portion of the mesh which remains exterior to the tadpole brain. FIG. 1 IE illustrates a stress-strain curve of PFPE-DMA film with 8 kDa molecular weight, the blue dash line indicates a linear relationship. FIG. 1 IF illustrates stretchability of SU-8 and PFPE-DMA films with 8 kDa molecular weight. FIG. 11G illustrates surface free energy of SU-8 and 8 kDa PFPE-DMA films. FIG. 11H illustrates contact angles of phospholipid (cell membrane analog) on SU-8 film, and of phospholipid on 8 kDa PFPE-DMA films. FIG. I ll illustrates photographs showing an embryo successfully implanted with a PFPE-DMA mesh. The dashed line circle highlights the portion of the mesh which remains exterior to the tadpole brain. FIG. 11 J illustrates schematics showing elongation of the neural tube during the embryo development of Xenopus laevis. The caudal region of the neural tube elongates to 3 times its initial length and forms the spinal cord while the cranial region elongates only 1.3 times its initial length and forms the brain. FIG. 1 IK illustrates schematics showing how anchors fix the stretchable mesh microelectronics to the substrate, keeping the neural plate properly positioned during neurulation for device internalization, and keeping the stretchable mesh electrode array attached to the neural plate. The device’s initial dimensions and stretchability allow the stage 15 embryo to be slid under the device for implantation. FIG. 1 IL illustrates the final design of the stretchable mesh microelectronics showing the architecture of the stretchable mesh electrode array, stretchable serpentine interconnects, anchors, stretchable ribbons, and blockers. The blocker prevents the mesh electrodes from implanting into the caudal region of the neural plate.

FIGs. 12A-12B illustrate mechanical simulation of stretchable mesh for brain implantation via embryo development. FIG. 12A illustrates snapshots of mechanical simulation of mesh-neural plate interaction (FIGs. 4 J, 4K), labeled with sequenced numbers. FIG. 12B illustrates snapshots of mechanical simulation procedure showing the stress distribution in the neural plates with and without stretchable mesh implanted. An embryo simulation without mesh implantation was used as a reference to calculate the additional stresses introduced by PFPE-DMA and SU-8 meshes.

FIGs. 13A-13H illustrate fabrication of PFPE-DMA encapsulated stretchable mesh microelectronics. FIGs. 13A and 13B illustrate schematics showing the structure (FIG. 13A) and section view (FIG. 13B) of the nitrogen chamber designed for use with the mask aligner in PFPE-DMA photopatteming. FIG. 13C illustrates a schematic showing how the nitrogen chamber is used with mask aligner. The mask aligner base fits the circular hole of the nitrogen chamber in (FIG. 13A). FIG. 13D illustrates microscopic BF images showing representative high-resolution PFPE-DMA photolithography patterns made with the nitrogen chamber. FIGs. 13E and 13F illustrate microscopic BF images showing the improved adhesion between gold interconnects and PFPE-DMA after inert gas plasma treatment. FIG. 13E, Without inert gas plasma treatment, aluminum/gold interconnects peel off from the PFPE-DMA film after Sputtering. FIG. 13F, With inert gas plasma treatment before sputtering, aluminum/gold interconnects strongly bond to the PFPE-DMA film. Dashed boxes highlight the sputtered regions on the PFPE-DMA films. FIG. 13G illustrates microscopic BF images showing the stretchable mesh electrode array region of PFPE-DMA device in fabrication steps corresponding to (FIG. 5C). Step 1 shows a homogeneous nickel layer. Step 2 is not included because the electrode array region does not have an SU-8 spacer. Step 3 shows platinum electrodes on the nickel layer. Electrodes are highlighted by red dashed circles. Steps 4-6 show sequential patterning of bottom PFPE-DMA, gold interconnects, and top PFPE-DMA layers. FIG. 13H illustrates schematics showing the postfabrication steps of PFPE-DMA encapsulated stretchable mesh microelectronics following (FIG. 5C). After nanofabrication, the device is soldered with a flexible flat cable (step 7) and bonded with a culture chamber (step 8). Then, the nickel layer is etched to release the device. Pt-black is electro-polymerized on electrodes to reduce electrode impedance. The device is washed with 0.1 x MMR and finally soaked in culture media (step 9).

FIGs. 14A-14O illustrate a characterization of PFPE-DMA encapsulated stretchable mesh microelectronics. FIGs. 14A and 14B illustrate scanning electron microscope (SEM) images showing top views (FIG. 14A) and perspective views (FIG. 14B) of the stretchable mesh electrode array portion of the PFPE-DMA device. Each layer is pseudo-colored and labeled. FIG. 14C illustrates a bright-field (BF) image of a PFPE-DMA encapsulated gold ribbon. FIG. 14D illustrates a atomic force microscopy (AFM) topography image of black box highlighted region in (FIG. 14C). (Right) height profiles of horizontal cross-sections highlighted in the left figure. FIG. 14E illustrates BF images showing wrinkles of (left) straight and (right) serpentine PFPE-DMA encapsulated gold interconnects. FIG. 14F illustrates a BF image of a gold interconnect without top PFPE-DMA passivation. FIG. 14G illustrates a AFM topography image of black box highlighted region in (FIG. 14F). (Right) Height profiles of horizontal cross-sections highlighted in the left figure. FIG. 14H illustrates a SEM image showing perspective views of the stretchable mesh electrode array portion of the PFPE-DMA device. FIG. 141 illustrates a SEM image of the dash-line box highlighted region in (FIG. 14H). FIG. 14J illustrates a SEM image showing cross-sections of PFPE- DMA encapsulated gold interconnects, along the dash-line in (FIG. 141). FIGs. 14H-14J, Each layer is pseudo-colored and labeled. FIG. 14K illustrates an electrode impedance at 1 kHz in 37 °C PBS of PFPE-DMA mesh electronics before and after stretching and bending. Bar plots indicate mean +/- s.e.m., each dot represents a single trial, two-tailed unpaired t- test, n = 4, ns, not significant. FIG. 14E illustrates a resistance as a function of strain during the transverse stretch test of PFPE-DMA encapsulated electronics. Bar plots indicate mean +/- s.e.m., each dot represents a single trial, two-tailed unpaired t-test, n = 4, ns, not significant. FIG. 14M illustrates an electrochemical impedance spectroscopy of electrodes in stretchable mesh electronics with and without Pt black coating. FIG. 14N illustrates an electrode impedance at 1 kHz in 37 °C PBS of PFPE-DMA mesh electronics fabricated in different batches. FIG. 140 illustrates a live cell ratio of wild-type rat cortical neurons after 10 days in vitro culture with PFPE-DMA mesh microelectronics, without any treatment (control), and with degradation products of PFPE-DMA. Bar plots indicate mean +/- s.e.m., each dot represents a single trial, two-tailed unpaired t-test, n = 6, ns, not significant.

FIGs. 15A-15E illustrate staining methods and extended immunofluorescence images. FIGs. 15A and 15B illustrate schematics showing the protocols for tissue clearing and wholemount staining (FIG. 15A) and cryosection staining (FIG. 15B) to characterize brain tissue implanted with stretchable mesh microelectronics. FIG. 15C illustrates a whole-mount- stained 3D reconstructed confocal fluorescence image of implanted mesh microelectronics showing that the mesh is embedded in the neural tissue. FIG. 15D illustrates a 3D reconstructed confocal fluorescence images of a whole-mount-stained cyborg tadpole whose device was implanted in the middle of neurulation. FIG. 15E illustrates confocal fluorescence images showing transverse sections of the fore-, mid-, and hindbrain of cyborg tadpoles fixed at 2-, 4- and 8-days post fertilization. In all images, 4',6-diamidino-2-phenylindole (DAPI) labels cell nuclei, acetylated-tubulin labels basal bodies, Rhodamine 6G (R6G) labels the device, and SRY-box transcription factor 2 (Sox2) labels neural stem cells. In fluorescence images of whole-mount staining samples, HuC/D labels neurons. In fluorescence image of cryosection staining sample, Mytl labels neurons.

FIGs. 16A-16L illustrate experimental setup, trajectory analysis, and examples of behavior tests. FIG. 16A illustrates schematics showing the setup for behavioral testing. In each test, a tadpole is placed in a clear tank, sitting on an upward-facing screen. The screen is programmed to display the appropriate stimulation pattern for the color preference, visual avoidance, and edge preference tests. In the color preference test, the screen alternated between displaying half white and half black for 40 seconds each. In the visual avoidance test, a black dot is directly controllable via a computer mouse. The operator moved the dot toward the tadpole. If the tadpole responded, the operator would proceed to initiate the next encounter. If the tadpole did not respond, the operator would initiate a new encounter after five seconds. In the edge preference test, the entire screen is white. The setup is placed inside a dark box to minimize light contamination. The interior of the box is coated black to minimize reflections from the screen. Tadpoles are recorded using a video camera pointed down on the tank through a hole in the top of the box. FIGs. 16B-16I illustrate time-lapse snapshots of a visual avoidance video showing the trajectory process of a behaving tadpole. The colored lines connect the position of the tadpole in adjacent frames to form a trajectory. Crosses are labeled in frames where the tadpole met the black dot. FIGs. 16J-16L illustrate representative traces of behavior test data (top) and corresponding analyzed data (bottom). FIG. 16 J, (Top) representative trajectories of tadpole movement in a color preference test. The green dotted lines indicate the boundary between the black and white areas. The right and left areas are white and black, respectively, from 0-40 s and switch colors from 40-80 s. Dashed and solid lines represent the trajectories of tadpole movement from 0-40 s and 40-80 s, respectively. (Bottom) Distance of the tadpoles to the middle line. FIG. 16K, (Top) representative trajectories of tadpole movement in a visual avoidance test. Green crosses indicate the locations where the tadpole encountered the black dots. (Bottom) Distance between the tadpole and the black dot during the test. FIG. 16L, (Top) representative trajectories of tadpoles in an edge preference test. The green ring indicates the outer quarter radius of the container defined as the edge in the experiment. (Bottom) Distance between the tadpole and the container center. The green color indicates the edge region. The statistic results of behavior tests, including examples FIG. 16J-16L, were presented in FIGs. -7O-7Q.

FIGs. 17A-17I illustrate an experimental setup, raw data, and reference comparison of continuous electrophysiology. FIG. 17A illustrates schematics showing the oxygen anesthetic system used to minimize tadpole movement during culture for recording. The system mixes the anesthetic media with fresh oxygen to minimize the effects of anesthesia on tadpole development. FIG. 17B illustrates schematics showing the recording setup for electrophysiological experiments. During recording, the culture chamber is placed in a Faraday cage on a grounded optic table. The I/O of the implanted mesh electronics is connected to a Blackrock recording system using a flexible flat cable (FFC) connector. A platinum probe is placed in the culture media as ground. FIG. 17C illustrates raw data of continuous recordings shown in (FIGS. 4A-4K). FIGs. 17D-17I illustrate reference comparison of continuous electrophysiology. Distribution plots showing comparisons of oscillation signal width (FIG. 17D) and interval); calcium-wave like signal width (FIG. 17F) and interval (FIG. 17G); and spike width (FIG. 17H) and interval (FIG. 171). Reference data is as follows: reference 154, reference 243, and reference 345. Reference 3 did not include the corresponding dataset for spike intervals, so it is not included in (FIG. 171). The results in (FIGs. 17D-17I) are determined from signals collected from three cyborg tadpoles.

FIGs. 18A-18T illustrate extended data and analysis of continuous electrophysiology. FIGs. 18A-18G illustrate drug tests of electrophysiology. FIGS. 18A and 18B illustrate drug test results of calcium-wave-like signals. FIG. 18A illustrates representative voltage traces from the cyborg tadpole under serial drug test conditions of no drug, cyanquixaline (CNQX)/[2R]-amino-5-phosphonopentanoate (APV), wash of CNQX/APV, and CNQX/APV/ Tetrodotoxin (TTX). FIG. 18B illustrates the change of wave number per minute during drug testing of calcium-wave-like signals. Bar plots indicate mean +/- s.d., each dot represents a recording trial. ***, p < 0.001, ****, p < 0.0001. FIGs. 18C-18G illustrate drug test results of spikes. FIG. 18C illustrates the change of firing rate during drug testing of spikes. The tadpole is treated with APV/CNQX, washed, then CNQX/APV/ TTX in series. Bar plots indicate mean +/- s.d., each dot represents a recording trial. *, p < 0.05, **, p < 0.01. FIG. 18D illustrates representative voltage traces from the cyborg tadpole under serial drug test conditions of no drug, bicuculline (BIC)/picro toxin (PTX), washed, then BIC/PTX/TTX. FIGs. 18E and 18F illustrate zoom-in views of the signal highlighted by green-I (FIG. 18E) and magenta-box (FIG. 18F) in (FIG. 18D). FIG. 18G illustrates the change of firing rate during drug testing of spikes. The tadpole is treated with BIC/PTX, washed, then BIC/PTX/TTX in series. Bar plots indicate mean +/- s.d., each dot represents a recording trial. *, p < 0.05. FIGs. 18H-18O illustrate a rorrelation of single-unit recording with electrode position, h, Representative voltage traces from a cyborg tadpole showing single-unit spikes. FIGs. 181 and 18J illustrate zoom-in views of the signal highlighted by green-box (FIG. 181) and magenta-box (FIG. 18 J) in (FIG. 18H). FIG. 18K, Average spike (mean +/- s.d.) sorted from the voltage traces in (FIG. 18H). FIGs. 18L-18O, Confocal fluorescence images of the cyborg tadpole brain slice showing channels of 4',6-diamidino-2- phenylindole (DAPI) (FIG. 18L), HuC/D (FIG. 18M), bright field (FIG. 18N) and composite (FIG. 180). The white dash circles highlight the position of the electrode, which recorded the single-unit spikes in (FIG. 18H). Electrode position was determined as described herein. FIG. 18P illustrates a correlation coefficient between channels of stage 24, 26, stage 40 local field potential signals and stage 40 spike signals. Positive correlation corresponds to a coefficient of 1, negative to -1, and no correlation to 0. White dots represent the lower quartile, median, upper quartile from bottom to top. Each translucent dot represents a sample. ****, p < 0.0001. FIG. 18Q illustrates a standard deviation of time delay between channels of stage 24, 26, and 40 local field potential signals. Lower standard deviations indicate greater synchronization between channels. Box plots indicate minimum, lower quartile, median, upper quartile, and maximum. Each white dot represents a sample. *, p < 0.1, ***, p < 0.001. FIGS. 18R-18T illustrate propagating wave signals in stage 20 embryonic brain, r, Schematic of the cyborg tadpole at developmental stage 20. FIG. 18S illustrates representative voltage traces from four channels in the cyborg tadpole at stage 20. FIG. 18T illustrates zoom-in views of the signals highlighted by dashed lines in (FIG. 18S).

FIGs. 19A-19I illustrate high density mesh electrode array enabling traces of single units. FIG. 19A illustrates schematics showing the electron beam fabrication of PFPE-DMA encapsulated stretchable mesh electronics with 32-channel tetrode-like electrode array. First, a nickel layer is deposited on a blank silicon oxide wafer as a sacrificial layer. A SU-8 layer is patterned as a spacer, platinum electrodes are photolithographically patterned (step 1). Then, the bottom PFPE-DMA (step 2), gold interconnects (step 3), and top PFPE-DMA layer (step 4) are lithographical patterned. The gold layer is pattern by electron beam lithography. PFPE-DMA layers are patterned by optical lithography. Two-level zoom in figures display the details of the mesh electrode arrays (highlighted in blue and red Boxes). FIG. 19B illustrates photographic images showing the free-floating 32-channel tetrode-like electrode array during stretching, bending, and twisting. FIG. 19C illustrates BF microscopic image showing the 128-channel tetrode-like electrode array for electrophysiological recording. FIG. 19D illustrates BF microscopic image of the zoom-in view of the red box highlighted region in (FIG. 19C). FIG. 19E illustrates BF microscopic image of the zoom-in view of the blue box highlighted region in (FIG. 19D). FIG. 19F illustrates BF microscopic image of the zoom-in view of the green box highlighted region in (FIG. 19E) showing the electrodes and stretchable interconnects. FIG. 19G illustrates single-unit waveform centroids (n =20 neurons) from continuous 5 days recording in a cyborg axolotl tadpole (centroid computed using spatial average across electrode positions weighted by the mean waveform amplitude at each electrode). Grey patterns indicate the positions and sizes of the mesh electrodes. FIG. 19H illustrates single-neuron waveform centroids throughout 5 days recording in a cyborg axolotl tadpole. Centroids for single neurons from the same day were labeled with the same color. Centroids for single neurons from subsequent days are connected with lines. Grey circles indicate the positions and sizes of the mesh electrodes. FIG. 191 illustrates average displacement of single-neuron centroids between different days. Grey contours indicate quintile boundaries of the distribution of centroid position displacement for the population.

FIGs. 20A-20C illustrate ISI, autocorrelation and waveform dynamics in axolotl recording. FIG. 20A illustrates inter-spike interval (ISI) of spikes sorted from axolotl recording. Each color corresponds to an identified single unit. FIG. 20B illustrates representative average single-unit waveforms at each of the recording electrodes over the course of 5 days recording in cyborg axolotl tadpole. Waveforms recorded from each day were plotted as a gradient color. FIG. 20C illustrates time evolution of the spike firing rate in cyborg axolotl embryo development and spine cutting. Bar plots indicate mean +/- s.d., each dot represents firing rate of a single unit.

FIGs. 21A-21E show single-unit action potential in axolotl embryonic brain development. FIG. 21A shows the representative average single-unit waveforms recorded from the same neurons overlaid with extremum electrodes over the 5-day recording period. FIG. 21B shows the temporal evolution of spike firing rates during axolotl embryonic development and following spinal cord injury and regeneration. Bar plots indicate mean ± s.d., with individual dots representing the firing rate of single units. FIG. 21C is the UMAP visualization of units sorted from the continuous recording of tail- amputated cyborg axolotl embryo. FIG. 21D shows firing rates of units recorded immediately before and after tail amputation of cyborg axolotl embryo. Bar plots indicate mean ± s.d., with individual dots representing firing rate of individual units, two-tailed unpaired t-test, ****, p < 0.0001. FIG. 21E shows normalized presence of units sorted from continuous recording of the tail- amputated axolotl embryo.

Face-down electrodes and input/output (I/O) pads without bottom passivation. Certain embodiments comprise face-down electrodes and input/output (I/O) pads without bottom passivation. When the device is holding an embryo and the mesh electrode array is attached to the neural plate, the face-down electrodes are used to establish seamless contact with the neural plate and allow signal recording (FIG. 22A). In previous SU-8 devices, I/O pads were fabricated directly on the bottom SU-8 passivation before being bonded to a connector (FIG. 22B). However, due to the soft nature of this PFPE-DMA device, it could not withstand the mechanical pressure during the bonding process, leading to the development I/O pads without bottom passivation (FIG. 22B).

Rationale behind sputtering deposition and choosing of 8 kDa PFPE-DMA. The introduction of the two new structures (the face-down electrodes and input/output (I/O) pads without bottom passivation) presented a challenge in fabrication, as the gold ribbons needed to cross the steps on the bottom PFPE-DMA passivation (FIG. 22A). To address this issue, sputtering was utilized instead of evaporation, to deposit gold on the top of the bottom PFPE- DMA. Unlike directional evaporation, nondirective sputtering allowed for gold deposition on the PFPE-DMA side walls, allowing it to cross the steps (FIG. 22B). However, sputtering brought a-other problem - the sputtered gold layer was difficult to lift off. These findings showed that a device made of softer PFPE-DMA had a lower overall lift-off yield, with yields of PFPE-DMA with molecular weights of 4-, 8-, 10-, and 12-kDa being around 95%, 90%, 70%, and 30% respectively. However, a softer device would also be more challenging to handle during implantation, as it would be harder to firmly position the embryo and ensure the electrode array attaches to the neural plate.

Implantation yield. Implantation can potentially fail due to several reasons: 1. When the vitelline membrane of the embryo was peeled off to expose the neural plate in a separate petri dish before transferring it to the culture chamber with the device, there was a chance that the embryo could burst if it came in contact with the airsurface interface while being transferred with the pipette. This precaution was taken to prevent device contamination if the peeling procedure was unsuccessful.

2. During the implantation process, the embryo was slid under the stretchable device using tweezers, with one tweezer holding the device in its stretched state while the other pushed the embryo underneath. There was a chance that the device could break if held too tightly.

3. If the implanted device was excessively rigid and adhesive, such as the SU-8 device, the embryo was at a significant risk of breaking during the implantation procedure.

4. Misalignment between the neural plate and the device could occur during implantation.

5. After implantation, the embryo's neurulation may fail.

6. After successful implantation, there is a possibility that the electrodes may not exhibit signals. For instance, in earlier versions of the device with face-up electrodes, the recording yield of signals by the electrodes was low.

By utilizing appropriate materials and structural design for the device, it is possible to significantly reduce the likelihood of encountering situations 3-6. However, situations 1 and 2 may still occur but are infrequent. Individual records of the most recent implantation procedure were documented (FIG. 42). Each embryo implantation has been labeled with viewpoints such as transferring, intact device, intact embryo, alignment, neurulation, survival, and recording, corresponding to situations 1-6. Embryos 1-8 were implanted with devices for recording, while embryos 9-20 were implanted with dummies for other tests. Based on the data from the table, the overall success rate of implantation was 80% (successful implantation cases over all cases), the overall device functionality rate was 78.57% (successful recording cases over all device cases), and the overall electrode functionality rate within the device was 88.36% (successful recording channels over all channels in device cases).

FIGs. 22A and 22B illustrate structures of PFPE-DMA embryo device. FIG. 19A illustrates schematics illustrating the face-up electrode and face-down electrode in the context of soft and stretchable mesh microelectronics for embryo implantation. FIG. 19B illustrates a schematic diagram of sputtering deposition (left) and evaporation deposition (right).

FIGs. 23A-23C illustrate long term rearing of cyborg tadpoles to cyborg frogs. FIG. 23A illustrates photos of a tadpole that develop to stage 60 and a tadpole that develop to stage 65. The dashed circles highlight the interconnects of mesh electronics outside the brain. FIG. 23B illustrates a survival rate of cyborg tadpole and control tadpole after long term rearing. FIG. 23C illustrates a development stage count of cyborg tadpole and control tadpole after long term rearing.

FIGs. 22A and 22B illustrate immuno staining images depicting the contact between tissues and mesh electronics. FIG. 22A illustrates a whole-mount-stained 3D reconstructed confocal fluorescence image of implanted mesh microelectronics showing that the mesh is embedded in the Neural tissue. FIG. 22B illustrates confocal fluorescence images showing transverse sections of the fore-, mid-, and hindbrain of cyborg tadpoles fixed at 3-, 4- and 8- days post fertilization. In all images, 4',6-diamidino-2-phenylindole (DAPI) labels cell nuclei, acetylated-tubulin labels basal bodies, R6G labels the device, and SRY-box transcription factor 2 (Sox2) labels neural stem cells. In fluorescence images of whole-mount staining samples, HuC/D labels neurons. In fluorescence image of cryosection staining sample, myelin transcription factor 1 (Mytl) labels neurons.

FIGs. 25A and 25B illustrate a quantitative analysis procedures of fluorescence images. FIG. 25 A illustrates cell counting in SRY-box transcription factor 2 (Sox2, neural stem cells) fluorescence images. First, the Sox2 image is binarized to identify the region of neuron stem cells (step 1). Then the binary Sox2 image is overlaid with the 4',6-diamidino-2- phenylindole (DAPI)-labeled cell nuclei to indicate the nucleus of neuron stem cells (step 2). Finally, the number of neuron stem cell nuclei was counted and reported (step 3). Counting method of myelin transcription factor 1 (Mytl, neurons) is the same. FIG. 25B illustrates fluorescent intensity quantifying of bromodeoxyuridine (BrdU) images. First, the DAPI image is binarized as the region of tissue (step 1). Then, the binary DAPI image was overlaid with the BrdU image (step 2). The fluorescent intensity of BrdU in DAPI tissue region was calculated and reported (step 3).

FIG. 26 illustrates a final design of the stretchable device with 32-channel tetrode-like mesh electrode array. The design contains a high-density mesh electrode array, stretchable serpentine interconnects, anchors, stretchable ribbons, and blockers. The blocker prevents the mesh electrodes from implanting into the caudal region of the neural plate.

FIGs. 27A-27F illustrate a neurulation implantation of PFPE-DMA mesh microelectronics in mouse embryos. FIGs. 27A-27C illustrate bright field (BF) microscopic images showing three mouse embryos were implanted with stretchable mesh microelectronics. FIG. 27D illustrates a confocal fluorescence image displaying transverse sections of a cyborg mouse embryo fixed at embryonic stage 16. In the image, cell nuclei are labeled in blue, and mesh electronics are labeled in red. FIG. 27E illustrates a BF microscopic image of the zoom-in view of the dash line box highlighted region in (FIG. 27D) showing the implanted mesh electronics. FIG. 27F illustrates a BF microscopic image of the zoom-in view of the dash line box highlighted region in (FIG. 27E) showing a serpentine unit.

FIGs. 28A-28C illustrate agarose fixation of cyborg frog tadpole. FIG. 28A illustrates front, perspective, and top views of 3D printing pieces for agarose fixation of cyborg tadpole. FIG. 28B illustrates Procedures of agarose fixation of cyborg tadpole. State 1, a layer of agarose is cured on the agar scaffold of the fixation apparatus. State 2, the fixation apparatus was put on top of the culture chamber containing the anesthetized cyborg tadpole. The design of the fixation apparatus ensures that the tadpole is automatically in the middle of the two agarose scaffolds when the apparatus is placed on top. State 3, a small amount of low melting point agarose fixes the tadpole with the scaffolds serving as anchor points. FIG. 28C illustrates Time-lapse photographic images demonstrating that the tail of the agarose-fixed tadpole is in motion.

FIGs. 29A-29F illustrate an electrophysiological recording of cyborg frog tadpole with 16-channel electrode array and agarose fixation. FIG. 29A illustrates time-lapse bright field microscopic images of a representative frog embryo implanted with 16-channel stretchable mesh electronics at different development stages showing the gradual internalization of the mesh electrode array (dashed lines) into the neural plate. FIG. 29B illustrates a raster plot of spikes sorted from recordings of a cyborg tadpole with and without agarose fixation. FIGS. 29C and 29D illustrate average waveforms (FIG. 29C) and interspike interval (ISI) (FIG. 29D) of spikes concat sorted from recordings in (FIG. 29B). Each color corresponds to an identified single unit. FIG. 29E illustrate a uniform manifold approximation and projection for dimension reduction (UMAP) analysis of neurons from recordings with and without agarose fixation. FIG. 29F illustrate representative average single-unit waveforms at each of electrodes from recordings with and without agarose fixation. Each color corresponds to an identified single unit.

EXAMPLE 3

1. Face-down electrodes. During implantation, the mesh was to be attached to an embryo, with its bottom side in contact with the neural plate (FIG. 33 A). Therefore, to allow signal recording, the electrodes were exposed on the bottom side of the electronics. A fabrication recipe was developed to expose the electrodes on the bottom surface of the mesh, ensuring direct contact with the neural plate. 2. I/O pad design. Conventional SU-8 electronics utilized SU-8 as a back layer for the I/O pad. However, PFPE-DMA, being too soft, would beak during flip-chip bonding if still used as the back layer. Therefore, the I/O pads were designed without the bottom passivation (FIG. 33B).

3. Choosing PFPE-DMA with a molecular weight of 8 kDa. The 8 kDa PFPE-DMA was selected based on the fabrication yield. To increase the adhesion between the Au layer and the PFPE-DMA layer during Au deposition. A new protocol was developed that treated the PFPE-DMA surface with inert gas plasma, and then used sputtering to deposit the Au layer. However, the sputtered Au layer was difficult to lift off. The findings showed that a device made of softer PFPE-DMA had a lower overall lift-off yield, with yields of PFPE- DMA with molecular weights of 4-, 8-, 10-, and 12-kDa being around 95%, 90%, 70%, and 30%, respectively. As the result, 8 kDa PFPE-DMA was selected, which had both decent softness and fabrication yield.

EXAMPLE 4

The following examples provide various embodiments of the invention. FIG. 35 shows immunostaining images depicting the contact between Xenopus brain tissues and mesh electronics. Whole-mount- stained 3D reconstructed confocal fluorescence images of implanted mesh microelectronics showing the mesh embedded in the neural tissue. 4', 6- diamidino-2-phenylindole (DAPI) labels cell nuclei, HuC/D labels neurons, and Rhodamine 6G (R6G) labels the device.

FIGs. 36A-36B show procedures for quantitative analysis of fluorescence images. FIG. 36A show cell counting in SRY-box transcription factor 2 (Sox2, neuron stem cells) fluorescence images. First, the Sox2 image is binarized to identify the region of neuron stem cells (step 1). Then, the binary Sox2 image is overlaid with the D API-labeled cell nuclei to indicate the nucleus of neuron stem cells (step 2). Finally, the number of neuron stem cell nuclei is counted and reported (step 3). FIG. 36B shows fluorescent intensity quantifying of bromodeoxyuridine (BrdU) images. First, the DAPI image is binarized to identify the tissue region (step 1). Then, the binary DAPI image is overlaid with the BrdU image (step 2). The fluorescent intensity of BrdU in the DAPI tissue region is calculated and reported (step 3).

FIG. 37 shows the design of the stretchable mesh electronics with a 32-channel mesh electrode array. The design contains a high-density mesh electrode array, stretchable anchors and ribbons, and blockers for embryo integration. FIG. 38A-38F show implantation of stretchable mesh electronics in mouse embryos. FIGs. 38A-38C are BF microscopic images showing three representative mouse embryos implanted with stretchable mesh electronics. FIG. 38D shows confocal fluorescence image showing coronal sections of a cyborg mouse embryo fixed at embryonic stage 16. Cell nuclei, blue, and mesh electronics, red (reflective mode). FIG. 38E show zoomed-in view of the white dashed box-highlighted region in (FIG. 38D) showing the 3D integration of mesh electronics with the brain. FIG. 38F shows a zoomed-in view of the white dashed box- highlighted region in (FIG. 38E) showing one stretchable ribbon.

FIGs. 39A-39J show implantation of stretchable mesh electronics in neonatal rat brain. FIG. 39A-39B are photographic images showing mesh electronics before (FIG. 39A) and after (FIG. 39B) implantation into a neonatal rat brain. FIGs. 39C-39D are photographic images showing the neonatal rat after stereotactic surgery (FIG. 39C) and after recovering from anesthetics (FIG. 39D). FIG. 39E shows representative filtered voltage traces (300- 3,000 Hz bandpass filter). FIG. 39F shows zoomed-in views of the signals highlighted by blue-, green- and red-dashed box-highlighted regions in (FIG. 39E). FIG. 39G shows a raster plot of single-unit action potentials sorted from the recording. FIGs. 39H-39J show principal component analysis (PCA) (FIG. 39H), average waveforms (mean ± s.d.) (FIG. 391) and ISI (FIG. 39 J) of two representative units.

FIGs. 40A-40H show neural recording in awake cyborg tadpole. FIG. 40A shows front, perspective, and top views of the 3D-printed holder for the head-fixed recording. FIG. 40B shows schematics showing the stepwise fixation of the cyborg tadpole for awake recording. Step 1: A layer of agarose is cured on the agar scaffold of the holder. Step 2: The holder is placed on top of the culture chamber containing the anesthetized cyborg tadpole. The design of the holder ensures that the tadpole fits in the middle of the two agarose scaffolds. Step 3: A small amount of low melting point agarose further fixes the tadpole with the scaffolds. FIG. 40C shows time-lapse photographic images showing that in this setting, the tail of the agarose-fixed tadpole can still move during recording. FIG. 40D shows a raster plot of spikes sorted from a representative recording of a cyborg tadpole with and without agarose fixation. FIGs. 40E-40F show average waveforms (FIG. 40E) and ISI (FIG. 40F) of spikes sorted from recordings in (FIG. 40D). FIG. 40G shows the uniform manifold approximation and projection (UMAP) analysis of neurons from recordings with and without agarose fixation. FIG. 40H shows representative average single-unit waveforms at each of the electrodes from recordings with and without agarose fixation. Each color corresponds to an identified unit. While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the disclosure includes that number not modified by the presence of the word “about.”

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMS What is claimed is:

1. A method, comprising: implanting a stretchable electronic component in a tissue in a juvenile organism, the electronic component defining at least a portion of an electrical circuit; and determining an electronic signal from the electronic component over a time interval of at least 2 weeks.

2. The method of claims 1, further comprising sensing a property around the stretchable electronic component, and producing the electronic signal based on the property.

3. The method of claim 2, wherein the property comprises an electronic property.

4. The method of any one of claims 2 or 3, wherein the property comprises a chemical property.

5. The method of any one of claims 2-4, wherein the property comprises a mechanical property.

6. The method of any one of claims 1-5, wherein the stretchable electronic component is configured to stretch in response to growth of the juvenile organism.

7. The method of any one of claims 1-6, wherein the stretchable electronic component is stretchable by at least 20% in a linear direction under a force of no more than 1 micronewton.

8. The method of any one of claims 1-7, wherein the juvenile organism is less than 1 week old.

9. The method of any one of claims 1-8, wherein the juvenile organism is a pre-puberty subject.

10. The method of any one of claims 1-9, comprising determining electronic signals from the stretchable electronic component for at least 4 weeks.

11. The method of any one of claims 1-10, comprising determining electronic signals from the stretchable electronic component for a period of time that is at least 5% of the average lifetime of the species to which the organism belongs.

12. The method of any one of claims 1-11, comprising determining electronic signals from the stretchable electronic component over a time interval of at least 4 weeks.

13. The method of any one of claims 1-12, wherein the tissue is a brain.

14. The method of any one of claims 1-12, wherein the tissue is a heart.

15. The method of any one of claims 1-14, wherein the organism is human.

16. The method of any one of claims 1-14, wherein the organism is non-human.

17. The method of claim 16, wherein the organism is a mouse.

18. A method, comprising: implanting a stretchable electronic component in a tissue in an organism using a guide; and removing the guide from the tissue without removing the electronic component.

19. The method of claim 18, wherein the organism is a juvenile organism.

20. The method of any one of claims 18 or 19, wherein the component is stretchable by at least 20% in a linear direction under a force of no more than 1 micronewton.

21. The method of any one of claims 18-20, wherein removing comprises physically removing the guide from the tissue.

22. The method of any one of claims 18-21, wherein removing comprises dissolving the guide within the tissue.

23. The method of any one of claims 18-22, wherein the guide comprises a planar substrate.

24. The method of any one of claims 18-23, wherein the guide comprises a tubular substrate.

25. The method of any one of claims 18-24, wherein the guide comprises a hydrogel.

26. The method of claim 25, wherein the hydrogel is frozen.

27. An article, comprising: a juvenile organism, implanted with a stretchable electronic component, the electronic component defining at least a portion of an electrical circuit.

28. Use of a stretchable electronic component for implantation into a juvenile organism.