Photo–cross-linkable, insulating silk fibroin for bioelectronics with enhanced cell affinity Jie Jua,b, Ning Hua,c, Dana M. Cairnsa, Haitao Liua,d, and Brian P. Timkoa,1 aDepartment of Biomedical Engineering, Tufts University, Medford, MA 02155; bKey Laboratory for Special Functional Materials, Ministry of Education, Henan University, Kaifeng 475004, China; cState Key Laboratory of Optoelectronic Materials and Technologies, School of Electronics and Information Technology, Guangdong Province Key Laboratory of Display Material and Technology, Sun Yat-Sen University, Guangzhou 510275, China; and dSchool of Materials Science and Technology, China University of Geosciences, Beijing 100083, China Edited by John A. Rogers, Northwestern University, Evanston, IL, and approved May 21, 2020 (received for review February 27, 2020) Bioelectronic scaffolds that support devices while promoting into the brain (19) or retina (20) of live animal models to achieve tissue integration could enable tissue hybrids with augmented chronically stable electrophysiological readouts. Collectively, these electronic capabilities. Here, we demonstrate a photo–cross-linkable studies demonstrate the extraordinary potential for highly flexible silk fibroin (PSF) derivative and investigate its structural, electrical, electronics for achieving 3D bioelectronic interfaces with a wide and chemical properties. Lithographically defined PSF films offered variety of tissues. tunable thickness and <1-μm spatial resolution and could be released Recent efforts have focused on materials that are not only from a relief layer yielding freestanding scaffolds with centimeter- biocompatible but also bioactive. These materials, which bond scale uniformity. These constructs were electrically insulating; multi- surrounding tissues through chemical or mechanical interactions, electrode arrays with PSF-passivated interconnects provided stable are commonly used with implants to achieve biointegration and electrophysiological readouts from HL-1 cardiac model cells, brain sli- mitigate potential fibrotic responses. One common example is ces, and hearts. Compared to SU8, a ubiquitous biomaterial, PSF hydroxyapatite, which is similar to bone and is frequently used as exhibited superior affinity toward neurons which we attribute to its a coating for metallic implants to promote osseointegration (21). favorable surface charge and enhanced attachment of poly-D-lysine Bioactive dielectrics could be especially relevant in bio- adhesion factors. This finding is of significant importance in bioelec- electronics, as they would promote seamless integration between tronics, where tight junctions between devices and cell membranes interconnects and the surrounding tissue. Bioactive materials are necessary for electronic communication. Collectively, our findings with tunable conductivity include glasses (22) and ceramics (23), ENGINEERING are generalizable to a variety of geometries, devices, and tissues, but these materials are often brittle and so are not ideal candi- establishing PSF as a promising bioelectronic platform. dates for flexible and stretchable electronics. Natural polymers represent an alternative approach, playing a central role in tissue silk fibroin | photo–cross-linkable | bioelectronics | cell affinity | scaffold engineering (24). One especially promising bioactive polymer is silk fibroin, derived from the cocoon of Bombyx mori. Silk ex- dvances in bioelectronics promise to blur the line between hibits tunable mechanical properties (25, 26), excellent bio- Aliving and artificial systems, enabling two-way communica- compatibility (27), and programmable biodegradability (28), and tion between those disparate but complementary components (1–3). Examples of bioelectronic devices include multielectrode Significance arrays (MEAs) and field-effect transistors (FETs) which have been interfaced with neuron or cardiac cells for multiplexed, Recent advances in bioelectronics have opened avenues to- real-time readouts of electrophysiological activity (4, 5), while ward hybrid engineered tissues with embedded devices that other classes of devices have achieved on-demand drug delivery, could monitor, modulate, or otherwise augment cellular func- localized stimulation, power generation, and chemical sensing tion. In order to achieve these “cyborgs,” however, both de- capabilities (6, 7). Advances in nanoscience have played a key vices and electronic interconnects must stably integrate with role in each of these areas by enabling cellular-scale, noninvasive the surrounding tissue. We investigated a photo–cross-linkable devices such as transparent graphene MEAs (8) and freestanding silk fibroin (PSF) derivative that could be selectively cross- nanowire or nanopillar arrays (9–11) that could access the cy- linked using conventional photolithography. We found that tosol for intracellular measurements. PSF was an ideal candidate for bioelectronic scaffolds: it was One important challenge in bioelectronics—particularly with electrically insulating and so could passivate electronic inter- regard to three-dimensional (3D) device configurations—relates connects, presented amenable surface chemistry to promote to the device substrate, which must support devices and inter- cellular adhesion, and could be realized as freestanding, three- connects, provide adequate electrical passivation to prevent dimensional constructs. PSF-based scaffolds are compatible short circuits, and be biocompatible. Organ-level studies have with a wide variety of devices and tissues, and so could enable been demonstrated using flexible, stretchable, and bioresorbable new classes of hybrid systems. materials (12–14). More recently, highly flexible, macroporous mesh electronics were demonstrated (15). Composed of devices Author contributions: J.J., N.H., D.M.C., H.L., and B.P.T. designed research; J.J., N.H., D.M.C., and H.L. performed research; J.J., N.H., D.M.C., H.L., and B.P.T. analyzed data; and metallic interconnects sandwiched between layers of SU8, a and J.J. and B.P.T. wrote the paper. – photo cross-linkable epoxy, mesh electronics functioned as de- The authors declare no competing interest. vice supports, electronic passivation, and tissue scaffold and have This article is a PNAS Direct Submission. been fabricated with tunable geometries using conventional Published under the PNAS license. photolithography processes. Significantly, they solved the prob- Data deposition: Raw data associated with Figs. 1–5 are available in the Harvard/Tufts lem of mechanical mismatches presented by conventional, rigid University Dataverse at https://dataverse.harvard.edu/dataset.xhtml?persistentId=doi:10. devices such as microwires and silicon-based microchips, thereby 7910/DVN/SXSI8O. enabling excellent 3D device integration within engineered or 1To whom correspondence may be addressed. Email: [email protected]. primary tissues with minimal immune response. They have been This article contains supporting information online at https://www.pnas.org/lookup/suppl/ implemented to realize synthetic bioelectronics-innervated cardiac doi:10.1073/pnas.2003696117/-/DCSupplemental. tissues (16, 17) and organoids (18) and have also been injected www.pnas.org/cgi/doi/10.1073/pnas.2003696117 PNAS Latest Articles | 1of8 Downloaded by guest on September 28, 2021 moreover has potential for chemical customization through re- While the PSF reserved most of the functional groups in native active amino acid side-chain groups (29). It has been demon- fibroin, additional chemical shifts in PSF spectra were ascribed to strated in a wide range of tissue models (30), including those that the IEM modification: 1.88 ppm (methyl group), 5.7 and 6.09 ppm incorporate cocultures and functional innervation (31). Bio- (vinyl group), 4.97 ppm (methylene bridge adjacent to O), and 3.27 electronic devices containing silk as a major dielectric compo- and 3.35 ppm (methylene bridge adjacent to N) (SI Appendix,Fig. nent would be complementary to existing tissue scaffolds, S1). We also investigated the influence of the methacrylate- potentially enhancing tissue/device integration. esterification reaction on the silk fibroin structure using attenu- Here, we describe a photo–cross-linkable silk fibroin (PSF) ated total reflectance Fourier-transform infrared spectroscopy (SI derivative and report how it may be used as a passivating di- Appendix,Fig.S2). PSF preserved characteristic crystalline peaks − − electric for bioelectronic devices. While photo–cross-linkable forms (1,621 and 1,701 cm 1) and random coil peak (1,641 cm 1) of native of silk fibroin have been demonstrated previously (32, 33), their silk fibroin (36), with the intensity of crystalline peaks higher and dielectric properties in bioelectronic systems have not been ex- random coil peak lower compared to native silk fibroin, indicating plored, nor has their ability to adhere delicate cell lines such as that PSF contained a higher fraction of crystalline domains. human induced neural stem cells (hiNSCs) (34) which have been integrated into 3D tissue cocultures and brain tissue models. We Characterization of Lithographically Defined Structures. After syn- achieved PSF via an isocyanate-hydroxyl reaction and demonstrated thesizing PSF we assessed its feasibility as a photoresist (Fig. 2A). a photoresist that was compatible with conventional photolithog- We chose Irgacure 2959 as the photoinitiator (PI) to trigger the raphy. We found that the thickness of the cross-linked PSF film cross-linking of PSF under