NATURE MATERIALS DOL: 10.1038/NMAT3404 ARTICLES network of 3D features that mimic the size scale and morphology Electronic system Biological system of submicron ECM features, such as the fibrous meshwork of brain Nanoscale ECM(ref. 26). Open mesh nanoES were made by photolithography with a regular structure, similar to the ECM of the ventricular myocardium"28.3D scaffolds were then realized in a straightfor 联y ward manner by directed mesh manipulation. The planar design and initial fabrication of these 3D ties developed for conventional planar nanoelectronics, and could enable integration of additional device components(for example, device number and overall scaffold size so that metal interconnects were stressed 8 1. Removal of the sacrificial layer prompted self-organization into three dimensions. Reconstructed 3D confocal fluorescence images of typical reticular Macroscale Nanoelectronics-tiss scaffolds viewed along the y and x axes(Fig. 2bI and II respectively) showed that the framework was 3D with a highly curvilinear and interconnected structure. The porosity (calculated from the initial Figure 1 Integrating nanoelectronics with cells and tissue Conventional planar device design and the final 3D construct volume)was bulk electronics are distinct from biological systems in composition, >99.8%, comparable to that of hydrogel biomaterials"-. Nanowire structural hierarchy, mechanics and function. Their electrical coupling at FEt devices( Fig. 2bII)within the scaffold spanned separations of the tissue /organ level is usually limited to the tissue surface, where only 7.3-324 um in three dimensions(Supplementary Fig S3), and the oundary or global information can be gleaned unless invasive approaches reticular scaffold heights were less than 300 um for our present are used. We have introduced a new concept by creating an integrated fabrication conditions. Devices can be made closer together(for system from discrete electronic and biological building blocks(for example, example, <0.5 um) by depositing nanowires more densely on semiconductor nanowires, molecular precursors of polymers and singl the substrate to improve the spatial resolution of nanoelectronic cells). Three biomimetic and bottom-up steps have been designed: step A, sensors; the span of device separations and scaffold heights can be patterning, metallization and epoxy passivation for single-nanowire FETs; increased substantially using larger field lithography(see below) step B, forming 3D nanowire FET matrices(nanoelectric scaffolds) by self Scanning electron microscopy (SEM) of the reticular nanoES manual organization and hybridization with traditional ECMs; step C,(Fig. 2c)revealed kinked nanowires(about 80 nm diameter ), and corporation of cells and growth of synthetic tissue through biological metallic interconnects (about 0. 7um width) contained within processes. Yellow dots: nanowire components; blue ribbons: metal and the SU-8 backbone (about 1 um width). The feature sizes are epoxy interconnects; green ribbons: traditional ECMs: pink: cells comparable to those of synthetic and natural ECMs (refs 3, 6), and are several orders of magnitude smaller than those for and to be highly flexible and biocompatible. NanoES were then electronic structures penetrating tissue in three dimensions. ombined with synthetic or natural macroporous ECMs providing The performance of devices was evaluated through water-gate ECMs with electrical sensory function and nanoES with biochem- measurements for the nanowire FET elements in the 3D scaffolds in ical environments suitable for tissue culture. Finally, cells were aqueous medium(Supplementary Information). The results show ultured within the nanoES (step C, Fig. 1)to yield 3D hybrid device yields(80%), conductances(1.52+0.61 uS; mean+. d nanoelectronics-tissue constructs. The emphasis on a nanoscale and sensitivities(8.07+2.92usV-)comparable to measurements and biomimetic bottom-up pathway allows minimally invasive from planar devices using similar nanowires 8 integration of electronic devices with cells and ECM components 3D mesh nanoES were realized by folding and rolling free at the subcellular level in three dimensions. The nanoES are standing device arrays. Mesh structures(Fig. 2all)were fabricated distinct from conventional 2D multi-electrode arrays, carbon such that the nanoES maintained an approximately planar nanotube/nanofibre arrays,, implantable micro-electrodesand configuration following relief from the substrate. A typical flexible/stretchable electrodes- in that the sensors are nanoscale 3.5 cm x 1.5 cm x 2 um mesh nanoES, was approximately semiconductors, and critically, that the sensor network is flexible, planar with 60 nanowire Fet devices in a regular array with cultures that are known to resemble the structure, function or is comparable to that of a honeycomb-like synthetic ECM physiology of living tissues. ngineered for cardiac tissue culture2. In addition, the nanowires We have designed two nanoES(Fig 2a)that are free-standing, (Fig. 2d1), metal interconnects(Fig. 2d2)and SU-8 structural flexible and contain similar components. Both were fabricated elements(Fig. 2d3) had an areal mass density of <60 ug cm-z on sacrificial layers, which were subsequently removed, yielding the lowest value reported so far for flexible electronics, which free-standing nanoES(Methods and Supplementary Figs SI and reflects our macroporous architecture. The mesh nanoES was S2). In brief, a layer of negative resist(SU-8)was coated on a nickel flexible and can be manually rolled into tubular constructs sacrificial layer, a solution with kinked or straight nanowires was with inner diameters at least as small as 1.5 mm( Fig. 2e),and deposited onto the SU-8 layer and allowed to evaporate, and then folded. Macroporous structures of the open mesh nanoES were U-8 was patterned by lithography to immobilize nanowires and formed either by loosely stacking adjacent mesh layers(Fig. 2f) to provide the basic framework for nanoES. Extra nanowires were or by shaping it with other biomaterials. These capabilities were washed away during the development process of the SU-8 structure. consistent with the estimated ultralow effective bending stiffness Metal contacts were patterned by lithography and deposition. (Supplementary Information), which was tuned between 0.006 Finally, a layer of SU-8 was deposited and lithographically defined and 1.3 nN m for this mesh and is comparable to recent planar as the upper passivation layer on the interconnects. epidermal electronics. Reticular nanoES were made by electron beam lithography The electrical transport characteristics of nanoES were (EBL). Self-organization( that is, folding according to the prede- evaluated in phosphate buffered saline. The fined layout of bending elements)created a random or regular 90-97%, with average device conductance 3 uS and sensitivity NatureMateriAlsIVol11iNovemBer2012Iwww.nature.com/naturematerial G 2012 Macmillan Publishers Limited. All rights reservedNATURE MATERIALS DOI:10.1038/NMAT3404 ARTICLES A B C Electronic system Nanoscale Nanowires Bottom-up Nanowire FET Nanoelectronic scaffold Nanoelectronics¬tissue hybrid construct Macroscale Biomimetics Biological system Figure 1 | Integrating nanoelectronics with cells and tissue. Conventional bulk electronics are distinct from biological systems in composition, structural hierarchy, mechanics and function. Their electrical coupling at the tissue/organ level is usually limited to the tissue surface, where only boundary or global information can be gleaned unless invasive approaches are used. We have introduced a new concept by creating an integrated system from discrete electronic and biological building blocks (for example, semiconductor nanowires, molecular precursors of polymers and single cells). Three biomimetic and bottom-up steps have been designed: step A, patterning, metallization and epoxy passivation for single-nanowire FETs; step B, forming 3D nanowire FET matrices (nanoelectric scaffolds) by self￾or manual organization and hybridization with traditional ECMs; step C, incorporation of cells and growth of synthetic tissue through biological processes. Yellow dots: nanowire components; blue ribbons: metal and epoxy interconnects; green ribbons: traditional ECMs; pink: cells. and to be highly flexible and biocompatible. NanoES were then combined with synthetic or natural macroporous ECMs providing ECMs with electrical sensory function and nanoES with biochem￾ical environments suitable for tissue culture. Finally, cells were cultured within the nanoES (step C, Fig. 1) to yield 3D hybrid nanoelectronics–tissue constructs. The emphasis on a nanoscale and biomimetic bottom-up pathway allows minimally invasive integration of electronic devices with cells and ECM components at the subcellular level in three dimensions. The nanoES are distinct from conventional 2D multi-electrode arrays23, carbon nanotube/nanofibre arrays24,25, implantable micro-electrodes23 and flexible/stretchable electrodes13–17 in that the sensors are nanoscale semiconductors, and critically, that the sensor network is flexible, macroporous and 3D. As a result, nanoES are suitable for 3D cell cultures that are known to resemble the structure, function or physiology of living tissues. We have designed two nanoES (Fig. 2a) that are free-standing, flexible and contain similar components. Both were fabricated on sacrificial layers, which were subsequently removed, yielding free-standing nanoES (Methods and Supplementary Figs S1 and S2). In brief, a layer of negative resist (SU-8) was coated on a nickel sacrificial layer, a solution with kinked or straight nanowires was deposited onto the SU-8 layer and allowed to evaporate, and then SU-8 was patterned by lithography to immobilize nanowires and to provide the basic framework for nanoES. Extra nanowires were washed away during the development process of the SU-8 structure. Metal contacts were patterned by lithography and deposition. Finally, a layer of SU-8 was deposited and lithographically defined as the upper passivation layer on the interconnects. Reticular nanoES were made by electron beam lithography (EBL). Self-organization (that is, folding according to the prede￾fined layout of bending elements) created a random or regular network of 3D features that mimic the size scale and morphology of submicron ECM features, such as the fibrous meshwork of brain ECM (ref. 26). Open mesh nanoES were made by photolithography with a regular structure, similar to the ECM of the ventricular myocardium27,28. 3D scaffolds were then realized in a straightfor￾ward manner by directed mesh manipulation. The planar design and initial fabrication of these 3D nanoES use existing capabili￾ties developed for conventional planar nanoelectronics, and could enable integration of additional device components (for example, memories and logic gates)29,30 and substantial increases in device number and overall scaffold size. The 2D structure of the reticular scaffold was designed so that metal interconnects were stressed18,31. Removal of the sacrificial layer prompted self-organization into three dimensions. Reconstructed 3D confocal fluorescence images of typical reticular scaffolds viewed along the y and x axes (Fig. 2bI and II respectively) showed that the framework was 3D with a highly curvilinear and interconnected structure. The porosity (calculated from the initial planar device design and the final 3D construct volume) was >99.8%, comparable to that of hydrogel biomaterials6–8 . Nanowire FET devices (Fig. 2bII) within the scaffold spanned separations of 7.3–324 µm in three dimensions (Supplementary Fig. S3), and the reticular scaffold heights were less than ∼300 µm for our present fabrication conditions. Devices can be made closer together (for example, < 0.5 µm) by depositing nanowires more densely on the substrate30 to improve the spatial resolution of nanoelectronic sensors; the span of device separations and scaffold heights can be increased substantially using larger field lithography (see below). Scanning electron microscopy (SEM) of the reticular nanoES (Fig. 2c) revealed kinked nanowires (about 80 nm diameter), and metallic interconnects (about 0.7 µm width) contained within the SU-8 backbone (about 1 µm width). The feature sizes are comparable to those of synthetic and natural ECMs (refs 3, 6), and are several orders of magnitude smaller than those for electronic structures23 penetrating tissue in three dimensions. The performance of devices was evaluated through water-gate measurements for the nanowire FET elements in the 3D scaffolds in aqueous medium (Supplementary Information). The results show device yields (∼80%), conductances (1.52±0.61 µS; mean±s.d.) and sensitivities (8.07±2.92 µS V−1 ) comparable to measurements from planar devices using similar nanowires18 . 3D mesh nanoES were realized by folding and rolling free￾standing device arrays. Mesh structures (Fig. 2aII) were fabricated such that the nanoES maintained an approximately planar configuration following relief from the substrate. A typical 3.5 cm × 1.5 cm × ∼ 2 µm mesh nanoES, was approximately planar with 60 nanowire FET devices in a regular array with a 2D open porosity of 75% (Fig. 2d). This mesh porosity is comparable to that of a honeycomb-like synthetic ECM engineered for cardiac tissue culture28. In addition, the nanowires (Fig. 2d1), metal interconnects (Fig. 2d2) and SU-8 structural elements (Fig. 2d3) had an areal mass density of <60 µg cm−2 , the lowest value reported so far for flexible electronics, which reflects our macroporous architecture. The mesh nanoES was flexible and can be manually rolled into tubular constructs with inner diameters at least as small as 1.5 mm (Fig. 2e), and folded. Macroporous structures of the open mesh nanoES were formed either by loosely stacking adjacent mesh layers (Fig. 2f) or by shaping it with other biomaterials. These capabilities were consistent with the estimated ultralow effective bending stiffness (Supplementary Information), which was tuned between 0.006 and 1.3 nN m for this mesh and is comparable to recent planar epidermal electronics17 . The electrical transport characteristics of the mesh nanoES were evaluated in phosphate buffered saline. The typical device yield is 90–97%, with average device conductance ∼3 µS and sensitivity NATURE MATERIALS | VOL 11 | NOVEMBER 2012 | www.nature.com/naturematerials 987 © 2012 Macmillan Publishers Limited. All rights reserved