复旦大学：《纳米线材料和功能器件》课程教学资料_纳米线-生物复合界面_Macroporous nanowire nanoelectronic scaffolds for synthetic tissues

ARTICLES nature PUBLISHED ONLINE: 26 AUGUST 2012 I DO1: 10. 1038/NMAT3404 materials Macroporous nanowire nanoelectronic scaffolds for synthetic tissues Bozhi tian, 2,3t, Jia Liut Tal Dvir2, 4f, Lihua jins Jonathan h. tsui2 Robert Langer, Daniel S Kohane 2* and Charles M. Lieber 5*,Quan Qing Zhigang Suo The development of three-dimensional (3D) synthetic biomaterials as structural and bioactive scaffolds is central to fields ranging from cellular biophysics to regenerative medicine. As of yet, these scaffolds cannot electrically probe the physicochemical ological microenvironments throughout their d macroporous interior, although this capability could have a marked impact in both electronics and biomaterials. here we address this challenge using macroporous, flexible and free-standing nanowire nanoelectronic scaffolds (nanoES), and their hybrids with synthetic or natural biomaterials. 3D macroporous nanoEs mimic the structure of natural tissue scaffolds, and they were formed by self-organization of coplanar reticular networks with built-in strain and by manipulation of 2D mesh matrices. NanoES exhibited robust electronic properties and have been used alone or combined with other biomaterials as biocompatible extracellular scaffolds for 3D culture of neurons, cardiomyocytes and smooth muscle cells. Furthermore, we show the integrated sensory capability of the nanoes by real-time monitoring of the local electrical activity within 3D nano ES/cardiomyocyte constructs, the response of 3D-nanoES-based neural and cardiac tissue models to drugs, and distinct ph changes inside and outside tubular vascular tooth muscle constructs ben lesign and functionalization of porous materials have network must have 3D interconnectivity and mechanical properties actively pursued to enable new material properties and similar to biomaterials applications-. In particular, the development of synthetic Here we introduce a conceptually new approach that meets this 3D macroporous biomaterials as extracellular matrices(ECMs) challenge by integrating nanoelectronics throughout biomaterials represents a key area because functionalized 3D biomaterials al- and synthetic tissues in three dimensions using macroporous low for studies of cell/tissue development in the presence of nanoelectronic scaffolds. We use silicon nanowire field-effect of the pharmacological response of cells within synthetic tissues capability for recording both extracellular and intracellular signals is expected to provide a more robust link to in vivo disease with subcellular resolution-. FET detectors respond to variations treatment than that from 2D cell cultures. Advancing fur- in potential at the surface of the transistor channel region, and ther such biomaterials requires capabilities for monitoring cells they are typically called active detectors. Metal-electrode222. throughout the 3D micro-environment. Although electrical sen- or carbon nanotube/nanofibre 2425-based passive detectors are not sors are attractive tools, it has not been possible to integrate considered in our work because impedance limitations(that is such elements with porous 3D scaffolds for localized real-time signal/noise and temporal resolution degrade as the onitoring of cellular activities and physicochemical change; metal or carbon electrodes is decreased)make it difficult to reduce such capability could lead to new lab-on-a-chip pharmacologi- the size of individual electrodes to the subcellular level -,a cal platforms.o and hybrid 3D electronics-tissue materials for size regime necessary to achieve a non-invasive 3D interface of synthetic biology electronics with cells in tissue Recently, there have been several reports describing the coupling Our approach(Fig. 1)involved stepwise incorporation of of electronics and tissues using flexible and/or stretchable planar biomimetic and biological elements into nanoelectronic networks devices-17 that conform to natural tissue surfaces. These planar across nanometre to centimetre size scales. First, chemically synthe- evices have been used to probe electrical activities near surfaces sized kinked or uniform silicon nanowires were de of the heart3-l5, brain 6 and skin".So far, seamless 3D integration randomly or in regular patterns for single-nanowire FETs-the of electronics with biomaterials and synthetic tissues has not nanoelectronic sensor elements of the hybrid biomaterials(step A, been achieved. Key points that must be addressed to achieve Fig. 1). Second, individual nanowire FEt devices were lithograph this goal include: the electronic structures must be macroporous, ically patterned and integrated into free-standing macroporous not planar, to enable 3D interpenetration with biomaterials; the scaffolds(step B, Fig. 1), the nanoES. The nanoES were designed electronic network should have nanometre to micrometre scale to mimic structures,and specifically, to be 3D, to have features comparable to biomaterial scaffolds; and the electronic nanometre to micrometre features with high(>99%)porosity 1 Department of Chemistry and Chemical Biology, Harvard University Cambridge Massachusetts 02138, USA, 2Department of Division of Critical Care Medicine, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115, USA, David HKoc tegrative Cancer Research, Massachusetts Institute of Technology, Cambridge Massachusetts 02139, USA, Department of Chemical Eng sachusetts Institute of Technology Cambridge Massachusetts 02139, USA, 'School of Engineering and Applied Sciences, Harvard Universit Massachusetts 02138, USA. These authors contributed equally to this work. ' e-mail: Daniel. ohane @childrens. harvard.edu; cml@cmliris harvardedu NATURE MATERIALS I VOL 11 I NOVEMBER 2012 I G 2012 Macmillan Publishers Limited All rights reserved

ARTICLES PUBLISHED ONLINE: 26 AUGUST 2012 | DOI:10.1038/NMAT3404 Macroporous nanowire nanoelectronic scaffolds for synthetic tissues Bozhi Tian1,2,3† , Jia Liu1† , Tal Dvir2,4† , Lihua Jin5 , Jonathan H. Tsui2 , Quan Qing1 , Zhigang Suo5 , Robert Langer3,4, Daniel S. Kohane2 * and Charles M. Lieber1,5* The development of three-dimensional (3D) synthetic biomaterials as structural and bioactive scaffolds is central to fields ranging from cellular biophysics to regenerative medicine. As of yet, these scaffolds cannot electrically probe the physicochemical and biological microenvironments throughout their 3D and macroporous interior, although this capability could have a marked impact in both electronics and biomaterials. Here, we address this challenge using macroporous, flexible and free-standing nanowire nanoelectronic scaffolds (nanoES), and their hybrids with synthetic or natural biomaterials. 3D macroporous nanoES mimic the structure of natural tissue scaffolds, and they were formed by self-organization of coplanar reticular networks with built-in strain and by manipulation of 2D mesh matrices. NanoES exhibited robust electronic properties and have been used alone or combined with other biomaterials as biocompatible extracellular scaffolds for 3D culture of neurons, cardiomyocytes and smooth muscle cells. Furthermore, we show the integrated sensory capability of the nanoES by real-time monitoring of the local electrical activity within 3D nanoES/cardiomyocyte constructs, the response of 3D-nanoES-based neural and cardiac tissue models to drugs, and distinct pH changes inside and outside tubular vascular smooth muscle constructs. T he design and functionalization of porous materials have been actively pursued to enable new material properties and applications1–3 . In particular, the development of synthetic 3D macroporous biomaterials as extracellular matrices (ECMs) represents a key area because functionalized 3D biomaterials al￾low for studies of cell/tissue development in the presence of spatiotemporal biochemical stimulants3–6 , and the understanding of the pharmacological response of cells within synthetic tissues is expected to provide a more robust link to in vivo disease treatment than that from 2D cell cultures6–8 . Advancing fur￾ther such biomaterials requires capabilities for monitoring cells throughout the 3D micro-environment6 . Although electrical sen￾sors are attractive tools, it has not been possible to integrate such elements with porous 3D scaffolds for localized real-time monitoring of cellular activities and physicochemical change; such capability could lead to new lab-on-a-chip pharmacologi￾cal platforms9,10 and hybrid 3D electronics–tissue materials for synthetic biology11,12 . Recently, there have been several reports describing the coupling of electronics and tissues using flexible and/or stretchable planar devices13–17 that conform to natural tissue surfaces. These planar devices have been used to probe electrical activities near surfaces of the heart13–15, brain16 and skin17. So far, seamless 3D integration of electronics with biomaterials and synthetic tissues has not been achieved. Key points that must be addressed to achieve this goal include: the electronic structures must be macroporous, not planar, to enable 3D interpenetration with biomaterials; the electronic network should have nanometre to micrometre scale features comparable to biomaterial scaffolds; and the electronic 1Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA, 2Department of Anesthesiology, Division of Critical Care Medicine, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115, USA, 3David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA, 4Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA, 5School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, USA. †These authors contributed equally to this work. *e-mail: Daniel.Kohane@childrens.harvard.edu; cml@cmliris.harvard.edu. network must have 3D interconnectivity and mechanical properties similar to biomaterials. Here we introduce a conceptually new approach that meets this challenge by integrating nanoelectronics throughout biomaterials and synthetic tissues in three dimensions using macroporous nanoelectronic scaffolds. We use silicon nanowire field-effect transistor (FET)-based nanoelectronic biomaterials, given their capability for recording both extracellular and intracellular signals with subcellular resolution18–21. FET detectors respond to variations in potential at the surface of the transistor channel region, and they are typically called active detectors21. Metal–electrode22,23 - or carbon nanotube/nanofibre24,25-based passive detectors are not considered in our work because impedance limitations (that is, signal/noise and temporal resolution degrade as the area of the metal or carbon electrodes is decreased) make it difficult to reduce the size of individual electrodes to the subcellular level21–23, a size regime necessary to achieve a non-invasive 3D interface of electronics with cells in tissue. Our approach (Fig. 1) involved stepwise incorporation of biomimetic and biological elements into nanoelectronic networks across nanometre to centimetre size scales. First, chemically synthe￾sized kinked18 or uniform silicon nanowires were deposited either randomly or in regular patterns for single-nanowire FETs—the nanoelectronic sensor elements of the hybrid biomaterials (step A, Fig. 1). Second, individual nanowire FET devices were lithograph￾ically patterned and integrated into free-standing macroporous scaffolds (step B, Fig. 1), the nanoES. The nanoES were designed to mimic ECM structures, and specifically, to be 3D, to have nanometre to micrometre features with high (>99%) porosity 986 NATURE MATERIALS | VOL 11 | NOVEMBER 2012 | www.nature.com/naturematerials © 2012 Macmillan Publishers Limited. All rights reserved

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 reserved

NATURE 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

ARTICLES NATURE MATERIALS DOl: 10.1038/NMAT3404 黝懿圃 N团 d下 Conductance (us) °。4。86 1234567891011121314 Number of turns Device index Figure 2 Macroporous and flexible nanowire nanoES. a, Device fabrication schematics. (D)Reticular nanowire FET devices. (ID) Mesh nanowire FET evices Light blue: silicon oxide substrates: blue: nickel sacrificial layers; green: nanoES; yellow dots: individual nanowire FETs. b, 3D reconstructed onfocal fluorescence micrographs of reticular nanoES viewed along the y(i)and x(ID)axes. The scaffold was labelled with rhodamine 6G. The overall size of the structure, x-y-z=300-400-200um. Solid and dashed open magenta squares indicate two nanowire FeT devices located on different planes along the x axis Scale bars, 20 um. c, SEM image of a single-kinked-nanowire FET within a reticular scaffold, showing (1)the kinked nanowire, (2)metallic interconnects(dashed magenta lines)and (3)the Su-8 backbone Scale bar, 2 um. d, Photograph of a mesh device, showing(1)nanowires, (2)metal lterconnects and ( 3)Su-8 structural elements. The circle indicates the position of a single- nanowire FET Scale bar, 2 mm. e, Photograph of a partially rolled-up mesh device Scale bar, 5mm. f, SEM image of a loosely packed mesh nanoES, showing the macroporous structure Scale bar, 100 um g, Histograms of nanowire FET conductance and sensitivity in one typical mesh nanoES. The conductance and sensitivity were measured in the water-gate configuration without rolling. The device yield for this mesh nanoES is 95% h, Water-gate sensitivity and conductance of a nanowire FET in a mesh device during the rolling Upper panel, schematic of the nanowire fet position(yellow dot) during the rolling process; 0-6 denote the number of turns. i, Relative change in conductance and sensitivity of 14 nanow s evenly distributed throughout a fully rolled-up mesh device. Upper panel, schematic 7uSv-(Fig. 2g). Representative conductance(G)data( Fig. 2h) and showed that the properties were approximately independent of from a single-nanowire FET( Fig. 2h, yellow dots, upper panel) location. Furthermore, 14 devices evenly distributed on six layers during the rolling process showed a <0 17 uS conductance change of a rolled-up scaffold( Fig. 2i) showed maximum AG=6.8% AG)or <2.3% total change for 6 revolutions. Device sensitivity and AS=6.9% versus the unrolled state, demonstrating device (S)remained stable with a maximum change(AS)of 0.031usv-, robustness. Repetitive rolling and relaxation to the flat state or 1.5% variation. The stable performance during rolling can be did not degrade the nanowire FET performance. These findings explained by the low estimated strains of metal (<0.005%)and SU-8 suggest the potential for reliable sensing/recording of dynamic and (<0. 27%)in this tubular construct(Supplementary Information), deformable systems NATURE MATERIALS I VOL 11 I NOVEMBER 2012 I G 2012 Macmillan Publishers Limited All rights reserved

ARTICLES NATURE MATERIALS DOI:10.1038/NMAT3404 x z y y z x de f 1 2 3 4 6 5 7 8 9 10 11 12 13 14 ~1.7 mm 1.5 mm 1 2 3 0 2 1 3 15 10 5 0 Count 10 5 0 Count Conductance (μS) gh i 0 2 4 6 8 4 6 8 10 12 b c I II 1 3 2 Sensitivity (μS V¬1) 9 8 7 2.3 2.1 1.9 0 1 2 3 4 5 6 Sensitivity (μS V¬1) Number of turns Conductance (μS) 10 5 0 ¬5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Device index ΔG/G, ΔS/S (%) a I II 4 5 6 Figure 2 | Macroporous and flexible nanowire nanoES. a, Device fabrication schematics. (I) Reticular nanowire FET devices. (II) Mesh nanowire FET devices. Light blue: silicon oxide substrates; blue: nickel sacrificial layers; green: nanoES; yellow dots: individual nanowire FETs. b, 3D reconstructed confocal fluorescence micrographs of reticular nanoES viewed along the y (I) and x (II) axes. The scaffold was labelled with rhodamine 6G. The overall size of the structure, x–y–z = 300–400–200 µm. Solid and dashed open magenta squares indicate two nanowire FET devices located on different planes along the x axis. Scale bars, 20 µm. c, SEM image of a single-kinked-nanowire FET within a reticular scaffold, showing (1) the kinked nanowire, (2) metallic interconnects (dashed magenta lines) and (3) the SU-8 backbone. Scale bar, 2 µm. d, Photograph of a mesh device, showing (1) nanowires, (2) metal interconnects and (3) SU-8 structural elements. The circle indicates the position of a single-nanowire FET. Scale bar, 2 mm. e, Photograph of a partially rolled-up mesh device. Scale bar, 5 mm. f, SEM image of a loosely packed mesh nanoES, showing the macroporous structure. Scale bar, 100 µm. g, Histograms of nanowire FET conductance and sensitivity in one typical mesh nanoES. The conductance and sensitivity were measured in the water-gate configuration without rolling. The device yield for this mesh nanoES is 95%. h, Water-gate sensitivity and conductance of a nanowire FET in a mesh device during the rolling process. Upper panel, schematic of the nanowire FET position (yellow dot) during the rolling process; 0–6 denote the number of turns. i, Relative change in conductance and sensitivity of 14 nanowire FETs evenly distributed throughout a fully rolled-up mesh device. Upper panel, schematic of the nanowire FET position (yellow dots). In h,i the thicknesses of the tubular structures have been exaggerated for schematic clarity. ∼7 µS V−1 (Fig. 2g). Representative conductance (G) data (Fig. 2h) from a single-nanowire FET (Fig. 2h, yellow dots, upper panel) during the rolling process showed a <0.17 µS conductance change (1G) or <2.3% total change for 6 revolutions. Device sensitivity (S) remained stable with a maximum change (1S) of 0.031 µS V−1 , or 1.5% variation. The stable performance during rolling can be explained by the low estimated strains of metal (<0.005%) and SU-8 (<0.27%) in this tubular construct (Supplementary Information), and showed that the properties were approximately independent of location. Furthermore, 14 devices evenly distributed on six layers of a rolled-up scaffold (Fig. 2i) showed maximum 1G = 6.8% and 1S = 6.9% versus the unrolled state, demonstrating device robustness. Repetitive rolling and relaxation to the flat state did not degrade the nanowire FET performance. These findings suggest the potential for reliable sensing/recording of dynamic and deformable systems. 988 NATURE MATERIALS | VOL 11 | NOVEMBER 2012 | www.nature.com/naturematerials © 2012 Macmillan Publishers Limited. All rights reserved

NATURE MATERIALS DOL: 10.1038/NMAT3404 ARTICLES 353pm 40μm 170um 20 SU-8 350 nm ejected length (um) / Figure 3 Geometry control by design in nanoES. a, b, Basic design and structural subunit for simulation. a, Top-down view of the entire subunit. Blue ribbons are stressed metal lines with SU-8 passivation. Red lines are single Su-8 ribbons without residual stress. b, Cross-sectional views of those two key structural elements used for simulation. c, Plot of projected (on the x-y plane) length versus height (in the z direction) for the vertical blue ribbon in a as determined from the simulation. Open red squares with error bars are experimental data(means +s.d. recorded in air for point a and B in a. The shows a 3D view of the simulated structure, and the scale bar shows different heights in the z direction. d, Schematic showing the integration of period v imulation of the bending of the subunit model for the reticular structure was carried out using the commercial finite element software l S Th reticular-device domains (light blue) into a flexible mesh (green ). In individual reticular domains the 3d device positions relative to the global flexible mesh can be controlled by their geometry designs (a-c). e, f, Design patterns ()and experimental data( ID) for two reticular units. SU-8, metal and nanowires are shown in blue, pink and yellow in e. Changing the structure of the connecting feature (white arrows) between adjacent device units during pattern design (I) yields controlled variations in the 3D positioning of the nanowire FETs, which can be further tuned by the stress in the metal connections. these experiments, the device positions are 40 um(ell) and 23 um(fiD) above the mesh plane Scale bars in e f, 20 um We have carried out simulations of a subunit of the reticular nano ES/collagen scaffold(Fig 4a)shows clearly that self-organizing reticular structure(Fig. 3a-c). Measurements of the collagen nanofibres(green)are fully entangled with the bending for the corresponding experimental structures(Fig 3c, nanoES, with no evidence of phase separation. SEM images of open red squchanges in structural parameters(for example, the lyophilization(Fig. 4b)show that the flexible nanoES mesh is res)are consistent with the simulation(Fig 3c). the open mesh nanoES/alginate hybrid scaffold produced by Additionally, total length of the subunit and thicknesses of SU-8 or metals) intimately anchored to the alginate framework, which has a yield predictable changes in the bending angle of the subunit similar pore structure as the pure alginate scaffold prepared (Supplementary Fig S4). In this way, ordered 3D nanowire FET under similar conditions. Finally, optical micrographs of a arrays can be designed and fabricated using reticular-or mesh-like multilayered mesh nanoESPLGA scaffold(Fig. 4c), which wa structures that incorporate multi-layer metal interconnects with prepared by electrospinning PLGA fibres on both sides of the built-in stress to self-organize(roll-up)the scaffold( Supplementary nanoES and subsequent folding of the hybrid structure, highlight Fig S4). Finally, we have designed reticular domains in mesh-like the intimate contact between nanoES mesh and PLGA fibres. structures(Fig. 3d). Images of reticular domains(Fig. 3e, f) show The hybrid nano ES/biomaterial 3D scaffolds retain the original that regular nanowire FET devices with distinct device positions nanowire Fet device characteristics. For example, measurements can be realized by varying the structural parameters of individual in 1 x phosphate buffered saline solution showed that AG/G elements. Overall, this approach yields hierarchical 3D nanoES with and AS/s were less than +9% for the mesh nanoES/PLGA ubmicrometre to micrometre scale control in reticular domains composite versus bare nanoES Hybrid nanoES were stable under and millimetre to centimetre scale in the mesh matrix by folding or cell culture conditions. For example, nanowire FET devices in rolling as shown above( Fig. 2). the hybrid reticular nanoES/Matrigel scaffold in neuron culture cular and mesh nanoES were also merged with media(Fig. 4d)had AS/S<+11% over a nine-week period conventional macroporous biomaterials. Specifically, gel casting, suggesting a capability for long-term culture and monitoring with lyophilization and electrospinning were used to deposit and the nanoES. These results show that nanoES can be combined construct macroporous collagen (Fig 4a), alginate ( Fig 4b) with conventional biomaterials to produce hybrid scaffolds that and poly(lactic-co-glycolic acid)(PLGA; Fig. 4c), respectively, now provide nanoscale electrical sensory components distributed around nanoES. A confocal fluorescence micrograph of a hybrid in three dimensions NatureMateriAlsIVol11iNovemBer2012Iwww.nature.com/naturematerial G 2012 Macmillan Publishers Limited. All rights reserved

NATURE MATERIALS DOI:10.1038/NMAT3404 ARTICLES A B 60 µm Free 40 µm -standing part 56 µm 170 µm fixed part SU-8 350 nm Cr 50 nm Pd 75 nm SU-8 350 nm SU-8 350 nm a b 40 30 20 10 0 0 20 40 60 80 A B Projected length (µm) Height (µm) 35.3 µm 0 c d ef I I II II y x Figure 3 | Geometry control by design in nanoES. a,b, Basic design and structural subunit for simulation. a, Top-down view of the entire subunit. Blue ribbons are stressed metal lines with SU-8 passivation. Red lines are single SU-8 ribbons without residual stress. b, Cross-sectional views of those two key structural elements used for simulation. c, Plot of projected (on the x–y plane) length versus height (in the z direction) for the vertical blue ribbon in a as determined from the simulation. Open red squares with error bars are experimental data (means ±s.d.) recorded in air for point A and B in a. The simulation of the bending of the subunit model for the reticular structure was carried out using the commercial finite element software ABAQUS. The inset shows a 3D view of the simulated structure, and the scale bar shows different heights in the z direction. d, Schematic showing the integration of periodic reticular-device domains (light blue) into a flexible mesh (green). In individual reticular domains, the 3D device positions relative to the global flexible mesh can be controlled by their geometry designs (a–c). e,f, Design patterns (I) and experimental data (II) for two reticular units. SU-8, metal and nanowires are shown in blue, pink and yellow in e. Changing the structure of the connecting feature (white arrows) between adjacent device units during pattern design (I) yields controlled variations in the 3D positioning of the nanowire FETs, which can be further tuned by the stress in the metal connections. In these experiments, the device positions are 40 µm (eII) and 23 µm (fII) above the mesh plane. Scale bars in e,f, 20 µm. We have carried out simulations of a subunit of the self-organizing reticular structure (Fig. 3a–c). Measurements of bending for the corresponding experimental structures (Fig. 3c, open red squares) are consistent with the simulation (Fig. 3c). Additionally, changes in structural parameters (for example, the total length of the subunit and thicknesses of SU-8 or metals) yield predictable changes in the bending angle of the subunit (Supplementary Fig. S4). In this way, ordered 3D nanowire FET arrays can be designed and fabricated using reticular- or mesh-like structures that incorporate multi-layer metal interconnects with built-in stress to self-organize (roll-up) the scaffold (Supplementary Fig. S4). Finally, we have designed reticular domains in mesh-like structures (Fig. 3d). Images of reticular domains (Fig. 3e,f) show that regular nanowire FET devices with distinct device positions can be realized by varying the structural parameters of individual elements. Overall, this approach yields hierarchical 3D nanoES with submicrometre to micrometre scale control in reticular domains and millimetre to centimetre scale in the mesh matrix by folding or rolling as shown above (Fig. 2). The reticular and mesh nanoES were also merged with conventional macroporous biomaterials. Specifically, gel casting, lyophilization and electrospinning were used to deposit and construct macroporous collagen (Fig. 4a), alginate (Fig. 4b) and poly(lactic-co-glycolic acid) (PLGA; Fig. 4c), respectively, around nanoES. A confocal fluorescence micrograph of a hybrid reticular nanoES/collagen scaffold (Fig. 4a) shows clearly that the collagen nanofibres (green) are fully entangled with the nanoES, with no evidence of phase separation. SEM images of the open mesh nanoES/alginate hybrid scaffold produced by lyophilization (Fig. 4b) show that the flexible nanoES mesh is intimately anchored to the alginate framework, which has a similar pore structure as the pure alginate scaffold prepared under similar conditions. Finally, optical micrographs of a multilayered mesh nanoES/PLGA scaffold (Fig. 4c), which was prepared by electrospinning PLGA fibres on both sides of the nanoES and subsequent folding of the hybrid structure, highlight the intimate contact between nanoES mesh and PLGA fibres. The hybrid nanoES/biomaterial 3D scaffolds retain the original nanowire FET device characteristics. For example, measurements in 1 × phosphate buffered saline solution showed that 1G/G and 1S/S were less than ±9% for the mesh nanoES/PLGA composite versus bare nanoES. Hybrid nanoES were stable under cell culture conditions. For example, nanowire FET devices in the hybrid reticular nanoES/Matrigel scaffold in neuron culture media (Fig. 4d) had 1S/S < ±11% over a nine-week period, suggesting a capability for long-term culture and monitoring with the nanoES. These results show that nanoES can be combined with conventional biomaterials to produce hybrid scaffolds that now provide nanoscale electrical sensory components distributed in three dimensions. NATURE MATERIALS | VOL 11 | NOVEMBER 2012 | www.nature.com/naturematerials 989 © 2012 Macmillan Publishers Limited. All rights reserved

ARTICLES NATURE MATERIALS DOl: 10.1038/NMAT3404 Figure 4 Hybrid macroporous nanoelectronic scaffolds. a, Confocal fluorescence micrograph of a hybrid reticular nanoES/collagen matrix. Green (fluorescein isothiocyanate): collagen type-I; orange(rhodamine 6G): epoxy ribbons. The white arrow marks the position of the nanowire. Scale bar, 10 um b, SEM images of a mesh nanoES/alginate scaffold, top (I) and side(ii)views. The epoxy ribbons from nanoES are false-coloured in brown for clarity. Scale bars, 200um(I)and 100 um(ID). c, A bright-field optical micrograph of the folded scaffold, showing multilayered structures of PLGa and nanoelectronic interconnects The inset shows a photograph of the hybrid sheet before folding. A sheet of PLGA fibres with diameters of 1-3 um was deposited on both sides of the device. No damage or reduction of device yield was observed following this deposition Scale bars, 200 um and 5mm(inset). d, Relative langes in nanowire Fet sensitivity over time in culture (37 C: 5%CO2, supplemented neurobasal medium). n=5: data are means +s d The hybrid nanoES were evaluated in 3D culture, 33 for several Extended studies will be needed to evaluate the nanoES cell types. Embryonic rat hippocampal neurons were cultured in the term implants, although the main component of nano for longer reticular nanoES/Matrigel for 7-21 days(Supplementary Fig S5). has demonstrated long-term chronic biocompatibility suitable for Reconstructed 3D confocal micrographs from a two-week culture in vivo recording" (Fig 5a, b and Supplementary Fig. S6)showed neurons with a high The monitoring capabilities of the nanoES were first demon- density of spatially interconnected neurites that penetrated the strated in a 3D cardiomyocyte mesh construct(Fig. 5g). The output reticular nanoES (Fig 5a), often passing through the ring structures recorded from a single-nanowire FET (Fig. 5g)200 um below the upporting individual nanowire FETs(Fig 5b and Supplementary construct surface showed regularly spaced spikes with a frequency Fig. S6). Notably, the widths of the scaffold elements(passivated of - 1 Hz, a calibrated potential change of -2-3 mv, a signal/noise metal interconnects and structural ribbons) were similar to those >3 and a 2 ms width. The peak amplitude, shape and widt of the neurite projections, demonstrating the combination of are consistent with extracellular recordings from cardiomyocytes. electronics with biological systems at an unprecedented similarity The potential of the nanoES-based 3D cardiac culture to monitor in scale. 3D nanoelectronic cardiac culture was achieved from appropriate pharmacological response was investigated by dosing hybrid mesh nano ES/PLGA scaffolds(Supplementary Figs S7-S9). the 3D cardiomyocyte mesh construct with noradrenaline(al Confocal fluorescence microscopy of a cardiac 3D culture(Fig. 5c) known as norepinephrine), a drug that stimulates cardiac con revealed a high density of cardiomyocytes in close contact with traction through Br-adrenergic receptors. Measurements from nanoES components. Epifluorescence micrographs of cardiac cells the same nanowire Fet device showed a twofold increase in th on the surface of the nanoES cardiac patch showed striations contraction frequency following drug application. Interestingly, characteristic of cardiac tissue,32( Fig. 5d and Supplementary recordings from two FETs from the cardiac patch on Figs S8 and S9). In addition, the in vitro cytotoxicity of nanoES in noradrenaline application showed submillisecond and millise 3D neural and cardiac culture was evaluated( Fig. 5e, f). Differences ond level, heterogeneous cellular responses to the drug (Sup Matrigel over 21 days, assessed with a standard LIvE/DEAd made with a reticular nanoES/neural construct ( Supplementary assay 5e), and between cardiac cells in hybrid mesh Fig. Sl1) showed that the 3D response of glutamate activation nanoES/Matrigel/PLGA and Matrigel/PLGA from 2 to 12 days, could be monitored. Together these experiments suggest nanoES measured with a metabolic activity assay(Fig. 5f), were minimal. constructs can monitor in vitro the response to drugs from 3D These studies show that on the 2-3 week timescale, the nanoes tissue models, and thus have potential as a platform for in vitro component of the scaffolds has little effect on the cell viability, and pharmacological studies Last, simultaneous recordings from thus can be exploited for a number of in vitro studies, including four nanowire FETs with separations up to 6.8 mm in a na drug screening assays with these synthetic neural and cardiac tissues. noES/cardiac construct( Fig. 5h) demonstrated multiplexed sensing NATURE MATERIALS I VOL 11 I NOVEMBER 2012 I G 2012 Macmillan Publishers Limited All rights reserved

ARTICLES NATURE MATERIALS DOI:10.1038/NMAT3404 a c b 1 23456789 ¬20 20 ¬10 10 0 d ΔS/S (%) Time (week) I II Figure 4 | Hybrid macroporous nanoelectronic scaffolds. a, Confocal fluorescence micrograph of a hybrid reticular nanoES/collagen matrix. Green (fluorescein isothiocyanate): collagen type-I; orange (rhodamine 6G): epoxy ribbons. The white arrow marks the position of the nanowire. Scale bar, 10 µm. b, SEM images of a mesh nanoES/alginate scaffold, top (I) and side (II) views. The epoxy ribbons from nanoES are false-coloured in brown for clarity. Scale bars, 200 µm (I) and 100 µm (II). c, A bright-field optical micrograph of the folded scaffold, showing multilayered structures of PLGA and nanoelectronic interconnects. The inset shows a photograph of the hybrid sheet before folding. A sheet of PLGA fibres with diameters of ∼1–3 µm was deposited on both sides of the device. No damage or reduction of device yield was observed following this deposition. Scale bars, 200 µm and 5 mm (inset). d, Relative changes in nanowire FET sensitivity over time in culture (37 ◦C; 5% CO2, supplemented neurobasal medium). n = 5; data are means ±s.d. The hybrid nanoES were evaluated in 3D culture32,33 for several cell types. Embryonic rat hippocampal neurons were cultured in the reticular nanoES/Matrigel for 7–21 days (Supplementary Fig. S5). Reconstructed 3D confocal micrographs from a two-week culture (Fig. 5a,b and Supplementary Fig. S6) showed neurons with a high density of spatially interconnected neurites that penetrated the reticular nanoES (Fig. 5a), often passing through the ring structures supporting individual nanowire FETs (Fig. 5b and Supplementary Fig. S6). Notably, the widths of the scaffold elements (passivated metal interconnects and structural ribbons) were similar to those of the neurite projections, demonstrating the combination of electronics with biological systems at an unprecedented similarity in scale. 3D nanoelectronic cardiac culture was achieved from hybrid mesh nanoES/PLGA scaffolds (Supplementary Figs S7–S9). Confocal fluorescence microscopy of a cardiac 3D culture (Fig. 5c) revealed a high density of cardiomyocytes in close contact with nanoES components. Epifluorescence micrographs of cardiac cells on the surface of the nanoES cardiac patch showed striations characteristic of cardiac tissue28,32 (Fig. 5d and Supplementary Figs S8 and S9). In addition, the in vitro cytotoxicity of nanoES in 3D neural and cardiac culture was evaluated (Fig. 5e,f). Differences between hippocampal neurons in reticular nanoES/Matrigel versus Matrigel over 21 days, assessed with a standard LIVE/DEAD cell assay33 (Fig. 5e), and between cardiac cells in hybrid mesh nanoES/Matrigel/PLGA and Matrigel/PLGA from 2 to 12 days, measured with a metabolic activity assay (Fig. 5f), were minimal. These studies show that on the 2–3 week timescale, the nanoES component of the scaffolds has little effect on the cell viability, and thus can be exploited for a number of in vitro studies, including drug screening assays with these synthetic neural and cardiac tissues. Extended studies will be needed to evaluate the nanoES for longer￾term implants, although the main component of nanoES, SU-8, has demonstrated long-term chronic biocompatibility suitable for in vivo recording34,35 . The monitoring capabilities of the nanoES were first demon￾strated in a 3D cardiomyocyte mesh construct (Fig. 5g). The output recorded from a single-nanowire FET (Fig. 5g) ∼200 µm below the construct surface showed regularly spaced spikes with a frequency of ∼1 Hz, a calibrated potential change of ∼2–3 mV, a signal/noise ≥3 and a ∼2 ms width. The peak amplitude, shape and width are consistent with extracellular recordings from cardiomyocytes20 . The potential of the nanoES-based 3D cardiac culture to monitor appropriate pharmacological response was investigated by dosing the 3D cardiomyocyte mesh construct with noradrenaline (also known as norepinephrine), a drug that stimulates cardiac con￾traction through β1-adrenergic receptors36. Measurements from the same nanowire FET device showed a twofold increase in the contraction frequency following drug application. Interestingly, recordings from two nanowire FETs from the cardiac patch on noradrenaline application showed submillisecond and millisec￾ond level, heterogeneous cellular responses to the drug (Sup￾plementary Fig. S10). Additionally, multiplexing measurements made with a reticular nanoES/neural construct (Supplementary Fig. S11) showed that the 3D response of glutamate activation could be monitored. Together these experiments suggest nanoES constructs can monitor in vitro the response to drugs from 3D tissue models, and thus have potential as a platform for in vitro pharmacological studies9,10. Last, simultaneous recordings from four nanowire FETs with separations up to 6.8 mm in a na￾noES/cardiac construct (Fig. 5h) demonstrated multiplexed sensing 990 NATURE MATERIALS | VOL 11 | NOVEMBER 2012 | www.nature.com/naturematerials © 2012 Macmillan Publishers Limited. All rights reserved

NATURE MATERIALS DOL: 10.1038/NMAT3404 ARTICLES cardiomyocyte ■ Control noradrenaline Figure 5 3D cell culture and electrical sensing in nanoES. a, b, 3D reconstructed confocal images of rat hippocampal neurons after a two -week culture in Matrigel on reticular nanoES. Red (Alexa Fluor 546): neuronal B-tubulin; yellow (rhodamine 6G): epoxy ribbons. The metal interconnects are false-coloured in blue, and are imaged in the reflected light mode. The white arrow highlights a neurite passing through a ring-like structure supporting a nanowire FET Dimensions in a, x: 317 um; y: 317 um; z: 100 um; in b, x: 127 um; y: 127 um:: z: 68 um. c, Confocal fluorescence micrographs of a synthetic cardiac patch (ll and lD), Zoomed-in view of the upper and lower dashed regions in l, showing metal interconnects, the SU-8 scaffold (arrows in ID)and electrospun PLGA fibres (arrows in IlD) Scale bar, 40 um. d, Epifluorescence micrograph of the surface of the cardiac patch. Green(Alexa Fluor 488) actin; blue(Hoechst 34580): cell nuclei. The position of the source-drain electrodes is outlined with dashed lines. Scale bar, 40 um. e, viable hippocampal neurons cultured in nanoES/Matrigel versus Matrigel. Cell viability was evaluated with a LIVE/DEAD cytotoxicity assay Cells were counted from 3D reconstructed confocal fluorescence micrographs n=6: data are means +s d Differences between groups were very small although statistically significant (p<0.05). f, Metabolic activity of cardiomyocytes evaluated using the MTs assay. n=6: data are means ts.d. Differences between and after(blue)applying noradrenaline. h, Multiplex electrical recording of extracellular field potentials from four nanowire FETs in a mesh nanoES. Dat groups were very small although statistically significant (p< 0.05). g, Conductance versus time traces recorded from a single-nanowire Fet before(black) are conductance versus time traces of a single spike recorded at each nanowire Fet. ith submillisecond time Increases in nanowire FET density, the use of cross-bar circuits and resolution. Our sign yields relatively sparse device implementing multiplexing/demultiplexing for addressing could distribution wi an area of about 3.5 x 1.5 cm. allow the nanoES scaffolds to map cardiac and other synthetic NatureMateriAlsIVol11iNovemBer2012Iwww.nature.com/naturematerial 991 G 2012 Macmillan Publishers Limited. All rights reserved

NATURE MATERIALS DOI:10.1038/NMAT3404 ARTICLES x y z Time (ms) 0 10 20 30 40 50 nS 20 nS Time 2 s Conductance Conductance + noradrenaline 100 50 0 7 14 21 2 4 6 8 10 12 1.5 1.0 0.5 0.0 x y z I II III Metal SU-8 PLGA a c d b e g h f Viable cell percentage (%) Neuron Time (day) Metabolic activity (a.u.) Time (day) Cardiomyocyte Control Sample Control Sample Figure 5 | 3D cell culture and electrical sensing in nanoES. a,b, 3D reconstructed confocal images of rat hippocampal neurons after a two-week culture in Matrigel on reticular nanoES. Red (Alexa Fluor 546): neuronal β-tubulin; yellow (rhodamine 6G): epoxy ribbons. The metal interconnects are false-coloured in blue, and are imaged in the reflected light mode. The white arrow highlights a neurite passing through a ring-like structure supporting a nanowire FET. Dimensions in a, x: 317 µm; y: 317 µm; z: 100 µm; in b, x: 127 µm; y: 127 µm; z: 68 µm. c, Confocal fluorescence micrographs of a synthetic cardiac patch. (II and III), Zoomed-in view of the upper and lower dashed regions in I, showing metal interconnects, the SU-8 scaffold (arrows in II) and electrospun PLGA fibres (arrows in III). Scale bar, 40 µm. d, Epifluorescence micrograph of the surface of the cardiac patch. Green (Alexa Fluor 488): α-actin; blue (Hoechst 34580): cell nuclei. The position of the source–drain electrodes is outlined with dashed lines. Scale bar, 40 µm. e, Percentage of viable hippocampal neurons cultured in nanoES/Matrigel versus Matrigel. Cell viability was evaluated with a LIVE/DEAD cytotoxicity assay. Cells were counted from 3D reconstructed confocal fluorescence micrographs. n = 6; data are means ±s.d. Differences between groups were very small although statistically significant (p < 0.05). f, Metabolic activity of cardiomyocytes evaluated using the MTS assay. n = 6; data are means ±s.d. Differences between groups were very small although statistically significant (p < 0.05). g, Conductance versus time traces recorded from a single-nanowire FET before (black) and after (blue) applying noradrenaline. h, Multiplex electrical recording of extracellular field potentials from four nanowire FETs in a mesh nanoES. Data are conductance versus time traces of a single spike recorded at each nanowire FET. of a coherently beating cardiac patch, with submillisecond time resolution. Our current device design yields relatively sparse device distribution with 60 devices over an area of about 3.5 × 1.5 cm2 . Increases in nanowire FET density, the use of cross-bar circuits and implementing multiplexing/demultiplexing for addressing30 could allow the nanoES scaffolds to map cardiac and other synthetic NATURE MATERIALS | VOL 11 | NOVEMBER 2012 | www.nature.com/naturematerials 991 © 2012 Macmillan Publishers Limited. All rights reserved

ARTICLES NATURE MATERIALS DOl: 10.1038/NMAT3404 Lumen Lumen Inner tubing PDMS Vascular construct Inner 1000 Time(s) Figure 6 Synthetic vascular construct enabled for sensing a, Schematic of the synthesis of smooth muscle nanoES. The upper panels are side views, and he lower ones are either top views (I and ii) or a zoom-in view (lll). Grey: mesh nano ES: blue fibres: collagenous matrix secreted by HASMcs; yellow dots nanowire FETs: pink: HASMCs. b, (I) Photograph of a single HASMC sheet cultured with sodium L-ascorbate on a nanoES (iI) Zoomed-in view of the dashed area in L, showing metallic interconnects macroscopically integrated with cellular sheet. Scale bar, 5 mm. c, Photograph of the vascular construct after rolling into a tube and maturation in a culture chamber for three weeks. Scale bar, 5 mm. d (i) Micro-computed tomograph of a tubular construct segment. (ID)Zoomed-in view of the area outlined in I. The arrows mark the individual nanowire FET-containing layers of the rolled construct. Scale bar, 1mm. e, Haematoxylin-Eosin-(I) and Masson-Trichrome-(Il; collagen is blue)stained sections(-6 um thick) cut perpendicular to the tube axis; lumen regions are labelled. The arrows mark the positions of Su-8 ribbons of the nanoES Scale bars, 50um f, Changes in conductance over time for two nanowire Fet devices located in the outermost (red) and innermost (blue) layers. The inset shows a schematic of the experimental set-up. Outer tubing delivered bathing solutions with varying pH (red dashed lines and arrows); inner tubing delivered solutions with fixed pH (blue dashed lines and arrows) 992 NATURE MATERIALS I VOL 11 I NOVEMBER 2012 I G 2012 Macmillan Publishers Limited All rights reserved

ARTICLES NATURE MATERIALS DOI:10.1038/NMAT3404 a b d f e c I II I II II 0 500 1,000 1,500 2,000 2,500 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 200 nS Outer Ag/AgCl tubing Inner tubing PDMS I II III Lumen Outer Inner Time (s) Conductance Lumen Lumen I Vascular construct Figure 6 | Synthetic vascular construct enabled for sensing. a, Schematic of the synthesis of smooth muscle nanoES. The upper panels are side views, and the lower ones are either top views (I and II) or a zoom-in view (III). Grey: mesh nanoES; blue fibres: collagenous matrix secreted by HASMCs; yellow dots: nanowire FETs; pink: HASMCs. b, (I) Photograph of a single HASMC sheet cultured with sodium L-ascorbate on a nanoES. (II) Zoomed-in view of the dashed area in I, showing metallic interconnects macroscopically integrated with cellular sheet. Scale bar, 5 mm. c, Photograph of the vascular construct after rolling into a tube and maturation in a culture chamber for three weeks. Scale bar, 5 mm. d (I) Micro-computed tomograph of a tubular construct segment. (II) Zoomed-in view of the area outlined in I. The arrows mark the individual nanowire FET-containing layers of the rolled construct. Scale bar, 1 mm. e, Haematoxylin–Eosin- (I) and Masson-Trichrome- (II; collagen is blue) stained sections (∼6 µm thick) cut perpendicular to the tube axis; lumen regions are labelled. The arrows mark the positions of SU-8 ribbons of the nanoES. Scale bars, 50 µm. f, Changes in conductance over time for two nanowire FET devices located in the outermost (red) and innermost (blue) layers. The inset shows a schematic of the experimental set-up. Outer tubing delivered bathing solutions with varying pH (red dashed lines and arrows); inner tubing delivered solutions with fixed pH (blue dashed lines and arrows). 992 NATURE MATERIALS | VOL 11 | NOVEMBER 2012 | www.nature.com/naturematerials © 2012 Macmillan Publishers Limited. All rights reserved

NATURE MATERIALS DOL: 10.1038/NMAT3404 ARTICLES tissue electrical activities over the entire constructs at high density The silicon substrate was then coated with a methyl methacrylate and poly(methyl in three dimensions We have also extended our approach towards the development ribbons and then non-symmetrical Cr/Pd/cr (1.5/50-80/50-80 nm) metals were of artificial tissue with embedded nanoelectronic sensory capa- interconnects. The bilities. Specifically, vascular nanoES constructs were prepared by stress, which drove 3D self-organization when the structure was relieved from blood vessels 7.3 except for the addition of the nanoES(Fig. 6a layer of SU-8, and EBL of sU- 8 followed by curing (180C, 20 min)was uy 3 processes analogous to those used for tissue-engineered autologous the substrate. The silico and Supplementary Fig. S12). Human aortic smooth muscle cells to define the SU-8 passivation layer over the deposited metal interconnects. The ascorbate to promote the deposition of natural ECM. The hybrid Transene Company, Danvers)for 60-120 min at 25C. Last, the free-standing nanoES/HASMC sheets( Fig. 6b) were rolled into multi-layer 3D nanoES were dried using a critical point dryer(Autosamdri 815 Series A, Tousimis) ubular structures and matured(Supplementary Information) stored in the dry state before use in tissue culture. Each EBL step will take without macroscopic delamination or desquamate nd analyses showed that the cells expressed smooth muscle and electron beam dosage(for example, the typical area dosages for SU S, a-actin(Supplementary Fig. S13), the key contractile protein in respvidual steps. A similar approach was used in the fabrication of the mesh nanoES ly). The entire fabrication took 2-5 days depending on the duration of the The distribution of nanoes in the tubular construct was visual-(Supplementary Information), except that p-type nanowires and photolithography ed by micro-computed tomography(HCT). A projection of the re- were used and the entire process took 2-3 da constructed 3D uCt data(Fig. 6d)revealed regularly spaced metal collagen or Matrigel solution onto the edge of(reticular nanoES)or directly above nterconnects with at least four revolutions(arrows, Fig. 6dIl), (mesh nanoES)the nanoES scaffolds, and at 4C. The solutions were consistent with the nanowire FET mesh and tissue rolling. Analyses allowed to form gels around nanoES under conditions of 37"C and 5%COz of haemotoxylin-Eosin- and Masson-trichrome-stained sections for at least 20 min. The 3D nanoES/alginate scaffolds were prepared from (Fig. 6e)revealed smooth muscle tissue 200 um thick, with elon- pharmaceutical-grade alginate(Protanal LF5/60, FMC Biopolymers)by calcium gated cells and collagenous nanofibres, and embedded su-8 ribbons gafoate crosmimkxng-jnod stubsexpot toprhlixrt na to es/PLGA brid scaife tion of nanowire FET nanoelectronics with healthy smooth muscle. the mesh nanoES. The hybrid scaffold can be folded to increase the thicknes The potential of this vascular construct to function as a al Sprague/Dawley biomedical device was demonstrated by 3D PH sensing(Fig. 6f, rat cardiomycytes and HASMCs were cultured in nanoES using stablished protocols(Supplementary Information). Optical micrographs of inset). As the extravascular pH was varied stepwise with luminal immunohistochemically and histologically stained samples were recorded using PH fixed, simultaneous recordings from p-type nanowire FETs either Olympus Fluoview FV1000 or O in the outermost layer showed stepwise conductance decreases nanoES T haracterized with Zeiss Ultra55/Supra55VP field-emission SEMs with a sensitivity of x32 mV per unit of pH. nanowire FETs in the HMXST micro-computed tomography X-ray imaging system(model: the innermost layer(closest to luminal)showed minor baselir HMXST225, X-Tek). The in vitro cytotoxicity of nanoES was evaluated using the fluctuations. This ability to resolve extravascular langes and the CellTiter 96 AQueous One Solution Cell Proliferation Assay(Promega makes possible the detection of inflammation, ischaemia, tumour Corporation). Cardiomyocyte recordings were carried out in Tyrode solution with micro-environments or other forms of metabolic acidosis due to a 100 mv d. c source for the nanowire FETs. The current lified with overproduction of organic acids or impaired renal acidification,40, multi-channel preamplifier, filtered with a 3 kHz low-pass filter (CyberA lantation of these nanoES-based and digitized at a 50kHz Axon Digi144 vascular and other nanoES-embedded constructs for in vivo studies used to deliver two eows s ngle polyd imethy siloxane microfluidic chamber will require substantial future work. tubings were used to deliver solutions with fixed and varied pH, respectively The nanoES concept and implementations described here rep- The electrical measurements were conducted using a lock-in resent a new direction in merging nanoelectronics with biological modulation frequency of z9 and 39Hz a time constant of nd an amplitude systems because we have demonstrated a 3D macroporous mate- rial/device platform that is distinct from either engineered tissue were used in all recording and sensing experiments. The calibrated potential or flexible electronics-72-. Looking forward, there are several between device conductance changes(in nanosiemens)and the sensitivities(in areas to develop. Cell interactions with nanoES could be tuned by microsiemens per volt or nanosiemens per volt)that are determined individually modification of the nanoES with growth determinants632 In addi- in water-gate experiments. tion,the elements in the nanoES could be expanded to incorporate nanoscale stimulators and stretchable designs to provide electrical Received 27 February 2012; accepted 19 July 2012 and mechanical stimulation to enhance cell culture published online 26 August 2012 Methods References Kinked and uniform silicon 1. Na, K. et al. Directing zeolite structures into hierarchically nanoporous nanocluster-catalysed methods described previously. Devices were fabricated on architectures. Science 333, 328-332(2011) ilicon substrates(Nova Electronic Mal 2. Schaedler, T.A. et al. Ultralight metallic microlattices. Science 334, Sio, or 100nm Sio2/200 nm Si, N, at the surface. EBL and photolithography 962-965(2011) on nickel relief layers were used to define the metal contacts to the nar 3. Place, ES. George, J H, williams, C.K.& Stevens, M. M. Synthetic polymer and the principal features of the scaffolds. The key steps used in the fabricatio scaffolds for tissue engineering. Chem. Soc. Rev. 38, 1139-1151( and deposited, and served as the relief layer for the free-standing scaffolds termed 4. Wylie, R G et al. Spatially controlled simultaneous patterning f the reticular nanoES were as follows. First, 100 nm nickel meta g a 300-500 nm layer of SU-8 photoresist (2000.5, MicroChem, Newton)wa 5. Kloxin, A M, Kasko, A M, Salinas, C N.& Anseth, K.S. Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science 324 -r* kinked nanowires was deposited onto the SU-8 layer After identifying imaging(Olympus BX51)and desig cal strategies terconnect and sU-8 patterns in IGOR Pro(Wave Metrics)and Design CAD, EBL for engineering complex tissues. Nature Nanotech. 6, 13-22(2011). was used to pattern the overall SU-8 scaffold structure around chosen nanowire 7. Krach P, Langer, R& Ferreira, L. Three-dimensional biomaterials hich was post-baked (65C and 95.C for 2 and 4 min, respectively)and cured for the study of human pluripotent stem cells. Nature Methods 8, (180C, 20 min) to yield the flexible structural support for metal interconnects. 731-736(2011) NatureMateriAlsIVol11iNovemBer2012Iwww.nature.com/naturematerial 993 G 2012 Macmillan Publishers Limited. All rights reserved

NATURE MATERIALS DOI:10.1038/NMAT3404 ARTICLES tissue electrical activities over the entire constructs at high density in three dimensions. We have also extended our approach towards the development of artificial tissue with embedded nanoelectronic sensory capa￾bilities. Specifically, vascular nanoES constructs were prepared by processes analogous to those used for tissue-engineered autologous blood vessels37,38 except for the addition of the nanoES (Fig. 6a and Supplementary Fig. S12). Human aortic smooth muscle cells (HASMCs) were cultured on 2D mesh nanoES with sodium ascorbate to promote the deposition of natural ECM. The hybrid nanoES/HASMC sheets (Fig. 6b) were rolled into multi-layer 3D tubular structures and matured (Supplementary Information) without macroscopic delamination or desquamation (Fig. 6c), and analyses showed that the cells expressed smooth muscle α-actin (Supplementary Fig. S13), the key contractile protein in smooth muscle37 . The distribution of nanoES in the tubular construct was visual￾ized by micro-computed tomography (µCT). A projection of the re￾constructed 3D µCT data (Fig. 6d) revealed regularly spaced metal interconnects with at least four revolutions (arrows, Fig. 6dII), consistent with the nanowire FET mesh and tissue rolling. Analyses of haemotoxylin–Eosin- and Masson-trichrome-stained sections (Fig. 6e) revealed smooth muscle tissue ∼200 µm thick, with elon￾gated cells and collagenous nanofibres, and embedded SU-8 ribbons from the nanoES (Fig. 6e). These findings confirm the 3D integra￾tion of nanowire FET nanoelectronics with healthy smooth muscle. The potential of this vascular construct to function as a biomedical device was demonstrated by 3D pH sensing (Fig. 6f, inset). As the extravascular pH was varied stepwise with luminal pH fixed, simultaneous recordings from p-type nanowire FETs in the outermost layer showed stepwise conductance decreases with a sensitivity of ∼32 mV per unit of pH. nanowire FETs in the innermost layer (closest to luminal) showed minor baseline fluctuations. This ability to resolve extravascular pH changes makes possible the detection of inflammation, ischaemia, tumour micro-environments or other forms of metabolic acidosis due to overproduction of organic acids or impaired renal acidification39,40 , although we stress that the implantation of these nanoES-based vascular and other nanoES-embedded constructs for in vivo studies will require substantial future work. The nanoES concept and implementations described here rep￾resent a new direction in merging nanoelectronics with biological systems because we have demonstrated a 3D macroporous mate￾rial/device platform that is distinct from either engineered tissue6,41 or flexible electronics13–17,42–44. Looking forward, there are several areas to develop. Cell interactions with nanoES could be tuned by modification of the nanoES with growth determinants6,32. In addi￾tion, the elements in the nanoES could be expanded to incorporate nanoscale stimulators and stretchable designs to provide electrical and mechanical stimulation to enhance cell culture. Methods Kinked and uniform silicon nanowires were synthesized by the nanocluster-catalysed methods described previously18. Devices were fabricated on silicon substrates (Nova Electronic Materials, n-type 0.005 V cm) with 600 nm SiO2 or 100 nm SiO2/200 nm Si3N4 at the surface. EBL and photolithography on nickel relief layers were used to define the metal contacts to the nanowires and the principal features of the scaffolds. The key steps used in the fabrication of the reticular nanoES were as follows. First, 100 nm nickel metal was patterned and deposited, and served as the relief layer for the free-standing scaffolds. Next, a 300–500 nm layer of SU-8 photoresist (2000.5, MicroChem, Newton) was deposited over the entire chip (Supplementary Fig. S1c) followed by pre-baking at 65 ◦C and 95 ◦C for 2 and 4 min, respectively; then an isopropanol solution of n +–n–n + kinked nanowires was deposited onto the SU-8 layer. After identifying nanowire positions by optical imaging (Olympus BX51) and designing the interconnect and SU-8 patterns in IGOR Pro (WaveMetrics) and DesignCAD, EBL was used to pattern the overall SU-8 scaffold structure around chosen nanowires, which was post-baked (65 ◦C and 95 ◦C for 2 and 4 min, respectively) and cured (180 ◦C, 20 min) to yield the flexible structural support for metal interconnects. The silicon substrate was then coated with a methyl methacrylate and poly(methyl methacrylate) double-layer resist, the resist was patterned over the chosen SU-8 ribbons and then non-symmetrical Cr/Pd/Cr (1.5/50–80/50–80 nm) metals were sequentially deposited followed by metal lift-off in acetone to form the nanowire interconnects. The non-symmetrical Cr/Pd/Cr layer structure yields a built-in stress, which drove 3D self-organization when the structure was relieved from the substrate. The silicon substrate was then coated with a uniform 300–400 nm layer of SU-8, and EBL of SU-8 followed by curing (180 ◦C, 20 min) was used to define the SU-8 passivation layer over the deposited metal interconnects. The reticular nanoES, including the interconnected kinked nanowire FET devices, were released from the substrate by etching of the nickel layer (Nickel Etchant TFB, Transene Company, Danvers) for 60–120 min at 25 ◦C. Last, the free-standing nanoES were dried using a critical point dryer (Autosamdri 815 Series A, Tousimis) and stored in the dry state before use in tissue culture. Each EBL step will take 10 min–2 h depending on the writing speed and area, feature size and complexity, and electron beam dosage (for example, the typical area dosages for SU-8 and poly(methyl methacrylate) EBL are 3–8 µC cm−2 and 500–1,000 µC cm−2 at 25 kV, respectively). The entire fabrication took 2–5 days depending on the duration of the individual steps. A similar approach was used in the fabrication of the mesh nanoES (Supplementary Information), except that p-type nanowires and photolithography were used and the entire process took 2–3 days. NanoES/collagen(Matrigel) hybrid matrices were made by casting 50∼2,000 µl collagen or Matrigel solution onto the edge of (reticular nanoES) or directly above (mesh nanoES) the nanoES scaffolds, and at ∼4 ◦C. The solutions were allowed to form gels around nanoES under conditions of 37 ◦C and 5% CO2 for at least 20 min. The 3D nanoES/alginate scaffolds were prepared from pharmaceutical-grade alginate (Protanal LF5/60, FMC Biopolymers) by calcium gluconate crosslinking and subsequent lyophilization to produce a sponge-like scaffold (5–15 mm×2–10 mm, d ×h). To prepare NanoES/PLGA hybrid scaffolds, a sheet of PLGA fibres with diameters of ∼1–3 µm was deposited on both sides of the mesh nanoES. The hybrid scaffold can be folded to increase the thickness. Embryonic Sprague/Dawley rat hippocampal cells, neonatal Sprague/Dawley rat cardiomycytes and HASMCs were cultured in nanoES using established protocols (Supplementary Information). Optical micrographs of immunohistochemically and histologically stained samples were recorded using either Olympus Fluoview FV1000 or Olympus FSX100 systems. The structures of nanoES were characterized with Zeiss Ultra55/Supra55VP field-emission SEMs or the HMXST micro-computed tomography X-ray imaging system (model: HMXST225, X-Tek). The in vitro cytotoxicity of nanoES was evaluated using the standard LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes, Invitrogen) and the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega Corporation). Cardiomyocyte recordings were carried out in Tyrode solution with a 100 mV d.c. source for the nanowire FETs. The current was amplified with a multi-channel preamplifier, filtered with a 3 kHz low-pass filter (CyberAmp 380) and digitized at a 50 kHz sampling rate18–20 (Axon Digi1440A). In extravascular pH sensing experiments, a single polydimethylsiloxane microfluidic chamber was used to deliver two flows of phosphate buffer solution, where inner and outer tubings were used to deliver solutions with fixed and varied pH, respectively. The electrical measurements were conducted using a lock-in amplifier with a modulation frequency of 79 and 39 Hz, a time constant of 30 ms and an amplitude of 30 mV; the d.c. source–drain potential is zero. Ag/AgCl reference electrodes were used in all recording and sensing experiments. The calibrated potential values (in millivolts) recorded from nanowire FETs were obtained as the ratios between device conductance changes (in nanosiemens) and the sensitivities (in microsiemens per volt or nanosiemens per volt) that are determined individually in water-gate experiments. Received 27 February 2012; accepted 19 July 2012; published online 26 August 2012 References 1. Na, K. et al. Directing zeolite structures into hierarchically nanoporous architectures. Science 333, 328–332 (2011). 2. Schaedler, T. A. et al. Ultralight metallic microlattices. Science 334, 962–965 (2011). 3. Place, E. S., George, J. H., Williams, C. K. & Stevens, M. M. Synthetic polymer scaffolds for tissue engineering. Chem. Soc. Rev. 38, 1139–1151 (2009). 4. Wylie, R. G. et al. Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels. Nature Mater. 10, 799–806 (2011). 5. Kloxin, A. M., Kasko, A. M., Salinas, C. N. & Anseth, K. S. 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ARTICLES NATURE MATERIALS DOl: 10.1038/NMAT3404 8. Hutmacher, D. w. Biomaterials offer cancer research the third dimension. 32. Sapir, Y, Kryukov, O. Cohen, S Integration of multiple cell-matrix ature mater9,9093(2010) nteractions into alginate scaffolds for promoting cardiac tissue regeneration. 9. Huh, D et al. Reconstituting organ-level lung functions on a chip. Science 328, materials32,1838-1847(201 1662-1668(2010) 33. Xu, T. et aL. Electrophysiological characterization of embryonic 3D collagen hydrogel Biomateria 11. Schwille, P Bottom-up synthetic biology: Engineering in a Tinkerer's world 30,4377-4383(2009) cience333,1252-1254(2011) 34. Cho, S.H. et al. Biocompatible SU-8-based microprobes for recording neural 12. Ruder, W. C. Lu, T. Collins, JJ Synthetic biology moving into the clinic. Tence333,12481252(2011) 1830-1836(2008) 13. Timko, B P. et al. Electrical recording from hearts with flexible nanowire 35. Voskerician, G et al. 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Christman, K L Decellularized brain matrix for cell culture and tissue engineering scaffolds. Tissu Author contributions EngA17,2583-2592(2011) B.T. J.L. T D, D.S.K. and C.M. L designed the ex ts B.T.and J. L perfo Hanley, P. J, Young, A. A, LeGrice, I. J, Edgar, S.G.& Loiselle, D S. Thre experiments. T.D. ]. T and Q.Q. assisted in the ini of the project. L J. and Z.S. dimensional configuration of perimysial collagen fibres in rat cardiac muscle performed calculations and simulations. B T, J.L. and C.M.L. wrote the paper. All ngths. Physio.517,831-837(1999 28. Engelmayr, G.C. -like honeycombs for tissue engineering of authors discussed the results and commented on the manuscript. cardiac anisotropy Nature Mater. 7, 1003-1010(2008) Additional information 841-850(2007) of the paper. Reprints and 30.Yan,h.etal.Programmablenanowirecircuitsfornanoprocessor.Nature470.permissionsinformationisavailableonlineatwww.nature.com/reprints.correspondence 240-2442011) and requests for materials should be addressed to D.S. K or C.M. 31. Wang, M. F, Maleki, T. Ziaie, B. Enhanced silicon microstructures via thermal shrinkage of a composite organic/inorganic Competing financial interests bilayer. IEEE/ASME J. Microelectromech. Syst. 17, 882-889(2008) The authors declare no competing financial interests. NATURE MATERIALS I VOL 11 I NOVEMBER 2012 I G 2012 Macmillan Publishers Limited All rights reserved

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Author contributions B.T., J.L., T.D., D.S.K. and C.M.L. designed the experiments. B.T. and J.L. performed experiments. T.D., J.T. and Q.Q. assisted in the initial stage of the project. L.J. and Z.S. performed calculations and simulations. B.T., J.L., D.S.K. and C.M.L. wrote the paper. All authors discussed the results and commented on the manuscript. Additional information Supplementary information is available in the online version of the paper. Reprints and permissions information is available online atwww.nature.com/reprints. Correspondence and requests for materials should be addressed to D.S.K. or C.M.L. Competing financial interests The authors declare no competing financial interests. 994 NATURE MATERIALS | VOL 11 | NOVEMBER 2012 | www.nature.com/naturematerials © 2012 Macmillan Publishers Limited. All rights reserved