Photo–cross-linkable, insulating silk fibroin for bioelectronics with enhanced affinity

Jie Jua,b, Ning Hua,c, Dana M. Cairnsa, Haitao Liua,d, and Brian P. Timkoa,1

aDepartment of , 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 (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 ultraviolet (UV) since Irgacure 2959 could be tuned over an order of magnitude and after exposure and is a high-efficacy PI and exhibits minimal toxicity to cells (PI developing exhibited features with ∼1-μm resolution, rivaling the refers to Irgacure 2959 in the following text other than special performance of SU8. We also found that PSF stably passivated note) (37, 38). We prepared solutions of PSF in HFIP at con- MEA interconnects, enabling bioelectronic readouts for up to 8 d centrations of 2, 4, 6, and 8% (wt/vol) with fixed PI concentration and stable passivation for at least 21 d. Significantly, hiNSCs at 0.4% (wt/vol), then spin-coated them on silicon substrates. demonstrated remarkably improved adhesion to PSF compared to After prebaking the substrates at 80 °C for 10–15 min and se- SU8, which we attribute to PSF’s favorable surface charge and lectively cross-linking the PSF by UV photolithography, we enhanced attachment of poly-D-lysine (PDL) adhesion factors. This postbaked the substrates for another 10–15 min at 80 °C and finding is of significant importance in bioelectronics, where tight then immersed them into a LiCl/DMSO solution (1 M) for junctions between devices and cell membranes are necessary for 0.5–2 min to remove the unreacted PSF. Thermogravimetric efficient electronic communication. These findings establish PSF as analysis and differential scanning calorimetry both revealed that a promising bioelectronic platform which could be extended to the glass transition of PSF was in a broad range, from about freestanding 3D configurations that could be stably integrated into 110 °C to over 170 °C, substantially higher than our pre- and awidevarietyoftissuemodels. postbake temperatures (SI Appendix, Fig. S3). We next assessed the suitability of PSF for achieving micro- Results scale structures by photolithography. First, we created a Tufts Synthesis of PSF. Fig. 1 outlines our synthetic route toward PSF. University logo pattern in which minimum feature sizes of ∼1 μm First, native silk fibroin was extracted from cocoons and freeze were clearly resolved (Fig. 2 B–D). Next, we demonstrated cross- dried as described previously (SI Appendix, Text)(35).Photo–cross- bar motifs similar to those achieved with mesh electronics (15). linkable methacrylate groups were then grafted onto pendant hy- Fig. 2E depicts the cross-sectional profile of the cross-bars along droxyl groups on the fibroin backbone by reaction with 2- the dashed red line, highlighting the sharp edges of the pattern. isocyanatoethyl methacrylate (IEM) in 60 °C dimethyl sulfoxide Significantly, height of the pattern (thickness of PSF film) could (DMSO) with 1 M lithium chloride and dibutyltin dilaurate catalyst be controlled by varying the concentration of PSF in HFIP so- under nitrogen atmosphere. The reaction product was thoroughly lution as well as the spin-coating speed (Fig. 2F), allowing us to rinsed with a mixture of acetone and ethanol, dried and lyophilized tune the thickness between ∼200 and 2,500 nm. We also found to form a PSF powder, then dissolved in hexafluoroisopropanol that PSF could be resolved over a very broad exposure window, (HFIP), to achieve a clear amber-colored solution. We confirmed which makes the processing time more flexible. For example, successful engraftment of the methacrylate groups by 1H NMR. films formed from 6% PSF solution and 2,000-rpm spin rate

Fibroin

Refinement

Cocoon

Sericin

O O

H H

O O O C O N C N OH OH O O

O Methacrylate OH

C Esterification

O O N

H O

Fig. 1. Synthetic route toward PSF. Native fibroin derived from cocoons was methacrylated through the hydroxyl groups on the chain to achieve PSF. After purification and freeze drying, PSF was readily dissolved in HFIP, yielding a clear amber-colored solution.

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.2003696117 Ju et al. Downloaded by guest on September 28, 2021 A UV Mask

Lithographyphy Developingping

PSF silicon wafer

B C D

10 2 100

1.8 E F2.5 G 1.6 2.0 1.4 1.5 200 1.2 1.0 1.0 Thickness (µm) Thickness (µm) 0.5 1.1 0.8 0 0 0.5 2468 4321 Concentration (%) Spin speed ( x103 rpm) 2 mm -0.1 Profile scanning

Fig. 2. Microfabrication with PSF. (A) Fabrication scheme using PSF as a negative photoresist, whereby an arbitrary pattern (e.g., Tufts logo) may be achieved by selective cross-linking of the PSF by conventional lithography followed by a developing step to wash away the unreacted material. (B) Optical image of a PSF Tufts logo pattern and (C and D) SEM expansions of regions in dotted boxes. (E) Optical image of PSF cross-bar pattern on glass substrate. (Inset) The curve

represents the height profile of structures shown in red dashed line. (F) PSF film thickness as a function of PSF photoresist solution concentration (n = 15) or ENGINEERING spin speed during spin coating (n = 4). (G) Photo of a highly flexible, freestanding PSF construct achieved through etching underlying zinc relief layer.

exposed at doses between 1,000 and 12,000 mJ/cm2 yielded insulating properties than the typically used dielectric layer of similar high-quality patterns after development (SI Appendix, sputtered 200-nm SiO2 films, which exhibited a ratio of only Fig. S4). In addition, the as-prepared PSF patterns were stable 2.79 times at 103 Hz. Moreover, we found that the insulating against common solvents frequently used in microfabrication and property of PSF was stable against PBS solution, with impedance at including isopropyl alcohol, ethanol, methanol, 1 kHz dropping by less than 20% over 21 d (SI Appendix,Fig.S7). DMSO, cell culture medium, and phosphate-buffered saline for We next assessed the ability of silk-passivated devices to form at least 1 mo (SI Appendix, Fig. S5), showing no evidence of bioelectronic interfaces with living cells or tissue. Our devices swelling or degradation. Finally, we demonstrated that our silk consisted of MEAs with circular 30-μm-diameter Au recording films could be released from an underling relief layer to achieve elements whose interconnects were passivated by 6% PSF films highly flexible, freestanding constructs that are amenable to mac- (Fig. 3 C–F). Fig. 3 C and D are a model and photograph of our roporous, conformable electronics with device arrangements in 3D MEA built on a printed circuit board (PCB) with a poly- configurations (Fig. 2G). Collectively, these features demonstrated dimethylsiloxane (PDMS) chamber adhered to enable cell cul- the suitability of PSF for bioelectronic applications in which stable, turing (SI Appendix, Text). We initially assessed our devices with high-resolution structures and flexible conformations, including HL-1 cardiac muscle models (40), which remained viable on our those in 3D, would be desirable. MEA substrates for at least 8 d in vitro (DIV). Fig. 3F shows a false-colored SEM image of the HL-1 monolayer at 4 DIV, Bioelectronic Interfaces with Silk-Based MEAs. We next explored the highlighting the cell (pink), recording element (light yellow), and feasibility of our silk-based structures to achieve stable bio- silk passivation layer (yellow). Throughout the culture period HL- electronic interfaces with cell culture medium and living cells. 1 monolayers beat spontaneously with stable firing intervals of Since bioelectronic devices are typically addressed by passivated 0.27 ± 0.01, 0.29 ± 0.02, and 0.26 ± 0.01 s at 4, 6, and 8 DIV, interconnects, we first assessed the dielectric properties of PSF respectively (n = 3). Fig. 3 G and H show extracellular recordings by electrical impedance spectroscopy. Gold working electrodes from a representative recording element at each of those time coated with dielectric films were submerged in PBS to mimic the points, showing an increase in magnitude over time—48.2 ± 0.9, conditions of a typical bioelectronic measurement. We then 218.3 ± 0.5, and 441.8 ± 0.6 μVat4,6,and8DIV,respectively— measured the impedance spectra across those films using a typ- consistent with an expected increase in cell–cell junctions and elec- ical three-electrode setup consisting of a Pt wire counter- trical synchrony throughout the culture over time (SI Appendix,Fig. electrode and Ag/AgCl reference electrode (Fig. 3 A and B and S8). Significantly, the filtered baseline noise was consistently less than SI Appendix, Text) (39). Films cast from 2% PSF demonstrated 4 μV across all time points, and the strongest bioelectronic interfaces poor passivation, potentially because of pinholes in the films, demonstrated signal-to-noise >78. which were evident under scanning electron microscopy (SEM) Our route toward silk-passivated bioelectronics represents a (SI Appendix,Fig.S6). In contrast, films cast from 4, 6, and 8% general platform that is readily extendable to a wide variety of PSF exhibited excellent dielectric properties over the range 10–106 applications. We anticipate that our devices will enable bio- Hz, with corresponding impedance amplitude ratio (|ZPSF,c|/ electronic interfaces with a wide variety of cell or tissue types; to 3 |Zbare_Au|) of 14.6, 17.2, and 16.6 at 10 Hz, for c = 4, 6, and 8% demonstrate this principle we achieved bioelectronic interfaces PSF, respectively. Significantly, these PSF films exhibited better with ex vivo heart and mouse brain. Female BALB/c mice were

Ju et al. PNAS Latest Articles | 3of8 Downloaded by guest on September 28, 2021 ABPotentiostat C D Electrode CEC RE WWEE PSF

Lead passivation PBS MEA InsulatedInsulated PDMS substrate Chamber

Gold lead

PSF layer

E GH4 DIV

6 DIV

Gold

PSF 50 µm 8 DIV F

0.1 s 10 µm

50 µv 0.5 s

Fig. 3. Bioelectronic measurements with PSF-based devices. (A) Electrical impedance measurement setup. (B) Impedance spectrum of PSF films spin-coated from 2 to 8% (wt/vol) HFIP solutions, compared to bare gold electrodes and electrodes coated with a silicon dioxide layer with thickness of 200 nm (n = 16). (C) Schematics of the MEA with interconnects passivated by PSF. The expansion illustrates a side view of a single device element and passivated interconnect. (D) Photograph of bioelectronic chip assembly including MEA, PDMS well, and PCB interface. (E) Optical image of a single device with interconnect passivated by PSF. (F) False-colored SEM image of HL-1 cultured on the PSF passivated device highlighting (bright yellow) gold layer, (deep yellow) PSF passivation layer,and (pink) single HL-1 cell. (G) Extracellular signal from a spontaneously beating HL-1 cell monolayer recorded on days 4, 6, and 8. (H) Magnified signals in the red dashed box in G.

killed at age of 6 wk, and the isolated hearts and brains were and SU8 cross-bars (Fig. 4I) to aid visualization. All of these immediately interfaced to silk-passivated MEA devices to record images show that hiNSCs distributed differently on the two types signals with signal-to-noise of 48 and 9.2, respectively (SI Ap- of surfaces: While hiNSCs appear both on the PSF cross-bars pendix, Fig. S9). In addition, we note that our silk films were and the interspaced glass substrates on the PSF patterned sur- highly transparent, with >90% transmission at visible wave- faces, they mostly avoided contact with the SU8 cross-bars on the lengths. This transmissivity was similar to SU8 films (41, 42) as patterned surfaces. Fig. 4 J and K are statistical analysis of the well as silk films that were not photo–cross-linked (13), opening fluorescence intensity of the different regions on the PSF- and avenues for simultaneous imaging and recording (SI Appendix, SU8-patterned surfaces. This quantitative comparison demon- Fig. S10). strates that the fluorescence intensity on the PSF cross-bars was similar to that on the glass substrate irrespective of statistics Characterization of Cell Adhesion. A crucial feature of bio- based on TUJ-1 staining (Fig. 4J) or DAPI staining (Fig. 4K). electronic scaffolds is that they must exhibit appropriate chem- However, the fluorescence intensity on the SU8 cross-bars was ical properties to form tight, seamless interfaces with the much lower than that on the interspaced glass substrates, in- surrounding cells. We studied this criterion using hiNSCs, which dicating fewer hiNSCs adhered to the SU8 cross-bars. This dis- spontaneously differentiated into neurons and have shown great tribution difference was even more apparent in the wide-field potential in engineered brain and other innervated tissue models imaging, as shown from the bright-field and fluorescent images (34, 43). For the purposes of these studies we examined two- in SI Appendix, Fig. S11, where the pristine glass wafer substrate dimensional systems, which enabled quantitative studies of cel- was used as the control surface. lular adhesion, migration, and surface chemistry. Since hiNSCs were plated on all surfaces at the same density We plated hiNSCs on PSF-patterned glass wafer surfaces and and cultured under identical conditions, we hypothesized that maintained their growth in culture medium (Materials and the final distribution behavior arose from differential cell affinity Methods). SU8-patterned glass wafers and pristine glass wafers to SU8 vs. PSF surfaces. To address this point, we assessed the were also seeded with hiNSCs at the same density as contrast and wettability of uniform PSF and SU8 films both before and after control groups. Immediately prior to plating the cells, all of the the PDL coating. These films were prepared in a fashion iden- surfaces were coated with PDL to promote hiNSC adhesion. tical to the cross-bar arrays presented in Fig. 4 and SI Appendix, After 5 d of culturing, the cells were fixed and stained with the Fig. S12, except that they were uniformly cross-linked by ex- neuron-specific marker β-III tubulin (TUJ-1) antibody and cluding the photomask during exposure. Before coating, the PSF DAPI. Fig. 4 A and B are, respectively, the bright-field images of and SU8 film showed water contact angles (WCA) of 48.1 ± 2.9° hiNSCs on the PSF- and SU8-patterned glass substrate. and 89.0 ± 2.3°, respectively (Fig. 5 A and B). After PDL coating Fig. 4 C–F are corresponding fluorescence images stained with at room temperature overnight, the WCA on the PSF film has TUJ1 antibody (DAPI). Fig. 4 G and H are the merged fluo- increased to 61.7 ± 1.5°, a change of approximate 13.6° although rescent images. The yellow dashed lines outline edges of the PSF still in the hydrophilic domain, indicating successful coating

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.2003696117 Ju et al. Downloaded by guest on September 28, 2021 PSF SU8 AB I

Crossbar Substrate

J C D ns

****

TUJ1 TUJ1

E F TUJ1 fluorescence intensity

PSF patterned SU8 patterned

K ****

DAPI DAPI GH ns ENGINEERING DAPI fluorescence intensity

100 µm 100 µm PSF patterned SU8 patterned

Fig. 4. hiNSC adhesion study on microfabricated PSF and SU8 structures. (A and B) Bright-field microscope images of cells plated on (Left) PSF- and (Right) SU8-patterned surfaces. The cells were stained with neuron-specific marker antibody TUJ1 (C and D) and DAPI (E and F), with G and H merged images for both surfaces. (I) Schematic of the patterned structure. (J and K) Statistical analysis of fluorescence intensity for TUJ1 and DAPI on PSF- and SU8-patterned surfaces, showing that cells distribute evenly on the PSF-patterned surfaces, while avoiding SU8 framework (****P < 0.0001; n = 7).

(Fig. 5C) (44), while the WCA on the SU8 decreased a little, to coated most and attracting the most cells, vice versa for the sit- 86.5 ± 1.8°, kept more hydrophobic than the postcoated PSF film uation of SU8 surfaces. (Fig. 5D). (n = 5 for all WCA measurements.) Since hydro- phobicity is adverse to cell adhesion and moderate hydrophilicity Discussion promotes cell adhesion, this difference in the wettability on both In this work we have investigated PSF, a photo–cross-linkable surfaces in turn affects cell distribution (45). derivative of silk fibroin which we achieved as a photoresist and To visualize the differential coating effect on PSF and SU8, we used to develop silk-based bioelectronic devices. While similar used fluorescein isothiocyanate (FITC)-labeled poly-L-lysine materials have been investigated previously, we specifically (PLL) to coat the PSF- and SU8-patterned surfaces and imaged assessed the unique properties of photo–cross-linkable silk as them under a fluorescent microscope. FITC-labeled PLL had the they related to bioelectronics. We demonstrated that PSF-based same coating mechanism as PDL on the substrate (physical ab- photoresists, compatible with standard photolithography, could sorption) but was also fluorescent (46). Fig. 5 E and F show the achieve patterned features with centimeter-scale uniformity and PSF-and SU8-patterned surfaces captured under the same ex- feature sizes with critical length < 1μm. This feature is important posing time. The PSF cross-bars are brighter than the inter- since it opens avenues for a wide variety of bioelectronic devices, spaced glass substrate, which is further brighter than the ranging from short channel-length FETs to organ-level struc- SU8 cross-bars. Fig. 5 G and H are the fluorescence intensity tures, including bioelectronics-embedded brain or cardiac tis- profile along the yellow dashed line in Fig. 5 E and F. For both sues. Moreover, we showed that our PSF exhibited not only surfaces, the interspaced glass substrates keep the same value of promising structural properties but also excellent electrical in- around 16, but the PSF cross-bars have a much higher value of sulating properties, which demonstrated that the material could nearly 90 and SU8 cross-bars have a lower value of around 10. be used both to support devices and also passivate electrical The different coating effect of PLL on the PSF, glass wafer, and interconnects. This is a crucial feature for a wide range of bio- SU8 surfaces can also be found in SI Appendix, Fig. S12 in wide- electronic devices, ranging from active FETs to passive MEAs (4). field imaging. All of these results confirmed that PLL was As proof-of-principle and to demonstrate the robustness of our physically absorbed more efficiently on the PSF surface com- technique, we demonstrated that MEAs with silk-passivated in- pared to SU8, leading to differing cell behaviors. The probable terconnects could record signals both from cultured cells (HL-1) reason for the different physical absorption is the varied surface as well as primary tissues (brain, heart). Significantly, in the case of electrical properties: PSF shows large negative charges in neutral HL-1 cells we recorded signals with signal-to-noise >78, which solution, glass surface is less negative charged, and SU8 is more compared favorably to other devices recently used to study cardiac prone to be neutral (47). When those surfaces are immersed in monolayers, including graphene MEAs with SU8 passivation (8). the positively charged PLL or PDL solution, the electrostatic A significant contribution of this study—and perhaps the one interaction is strongest on the PSF surfaces, leading to being that most distinguishes it from recent works involving SU8—is

Ju et al. PNAS Latest Articles | 5of8 Downloaded by guest on September 28, 2021 3.0 ± 0.1 and 4.4 ± 0.2 GPa (49). We note that the mechanical CA = 89.0±2.3º ABCA = 48.1±2.9º properties of PSF could be further tuned through 1) molecular weight at the point of fibroin extraction from silk fibers, 2) methacrylate or other ligand content during chemical modi- fication, or 3) photoinitiator concentration (50). Additionally, silk fibroin thin films had a reported tensile strength of ∼100 MPa, which is about three times greater than other polymer PSF-before coating SU8 - before coating systems including SU8 (50, 51). This property is especially de- sirable for flexible electronics and in load-bearing applications, CDCA = 61.7±1.5º CA = 86.5±1.8º e.g., bioelectronics-embedded cardiac tissues. Finally, the adhe- sion strength between PSF and gold is an important consider- ation. Because of its amino and carboxylate groups, silk fibroin adhered tightly to gold and moreover has been used as an ad- hesion layer between gold and silicon oxide for optically active devices (52). Since the amino groups in PSF are unreacted, we expect good adhesion with our systems as well; we have verified PSF-after coating SU8 -after coating this point experimentally through our long-term silicon oxide adhesion (SI Appendix, Fig. S5) and impedance (SI Appendix, EF Fig. S7) studies. Taken together, our results demonstrate that PSF is a prom- ising route toward bioelectronic scaffolds, representing a general platform that could be applied to a wide variety of cell and tissue types. The functionality of PSF described here could be further extended through covalently attached ligands, e.g., to achieve integrin-specific binding, direct neurite outgrowths, or achieve 100 µm 100 µm sustained drug release capabilities (53). Moreover, we note that silk fibroin bioinks are compatible with 3D printing processes G H (54), which in conjunction with techniques to print flexible and .) a.u.) .u stretchable circuits (55) could enable complex bioelectronic sity ( sity (a

en scaffolds with geometry tailored to the specific application. t n ten Taken together, the advantages of PSF could enable new classes ce i ce in

en of hybrid tissues with seamlessly integrated bioelectronics that scen re

resc monitor, modulate, or otherwise augment tissue function. uo uo Fl l F 0 300 600 900 1200 1500 0 300 600 900 1200 1500 Materials and Methods Distance (µm) Distance (µm) Synthesis of PSF. Silk fibroin refinement procedures are described in SI Ap- Fig. 5. (A–D) Contact angle measurements on PSF (A and C)orSU8(B and pendix. We dried 1.6 g lyophilized silk fibroin in a 60 °C oven overnight prior D) films either with or without PDL coating. (E and F) Fluorescence micros- to dissolving in 160 mL 1 M LiCl/DMSO solution to achieve a final concen- copy of FITC-PLL coated glass surfaces patterned with PSF or SU8 cross-bars. tration of 1% (wt/vol). The mixture was maintained at 60 °C with continuous (G and H) Profile of the fluorescence intensity along the dashed yellow line stirring and degassing via nitrogen purge. After the solution became clear in PSF- and SU8-patterned glass substrate, respectively. (∼20 min), 3.18 mL IEM and 60 μl dibutyltin dilaurate (DBTDL) were added into the solution quickly. The reaction was allowed to proceed for 5 h before pouring into a mixture of cold acetone and ethanol (volume ratio of 1:1) to precipitate the white flocculent postmodified fibroin. The product was our observation that PSF exhibited substantially improved rinsed 3× with acetone/ethanol mixture, and washed 2× with deionized properties for cell culture: It was more hydrophilic in its as- water, then lyophilized for 1–2 d, yielding dried and purified PSF (32, 35). prepared state, more effectively bound the poly-D-lysine adhe- sion factor, and at least in the case of hiNSC-derived neurons far HL-1 Culturing and Fixation for SEM. more effectively promoted soma adhesion as well as neurite Culture. HL-1 cells were plated at a density of 2 × 105 cells per cm2 on the extension. While highly flexible SU8 scaffolds have demon- fibronectin precoated flask and MEA surfaces and maintained in the Clay- strated remarkable promise in bioelectronics, providing stable comb medium supplemented with 10% fetal bovine serum, 2 mM L-gluta- − recordings from brain or retina with minimal immune response, mine, 0.1 mM norepinephrine, and 100 U ml 1 penicillin and 100 mg/mL these studies have been largely confined to mature cells in vivo. streptomycin in a cell-culturing incubator. Medium was changed daily. SEM studies. The few demonstrations of SU8 in engineered tissue systems re- HL-1 cells were fixed with 2% glutaraldehyde at 4 °C for 1 h, then dehydrated with a gradient series of ethanol at 50, 60, 70, 80, 90, and quired a chemically distinct matrix [e.g., coated poly (lactic- 100% concentrations (vol/vol), 10 min for each concentration. Afterward, coglycolic acid) fibers] to promote 3D adhesion and spreading. the sample was dried using a liquid CO2 critical point dryer. Prior to SEM, The ability of PSF to promote adhesion is likely to improve cou- samples were sputter coated with a 10-nm gold layer to impart conductivity pling between cell membranes and devices, thereby improving (11, 56). signal-to-noise (48). This property is especially apropos within the context of nerve tissue engineering where neurons are relatively hiNSC Culturing and Immunohistochemistry. sparse compared to cardiac tissue. Since 3D models for brain and Culture. hiNSC lines derived from adult human adipose stem cells were other innervated tissues have been achieved in silk scaffolds (30, generated as previously described. Surfaces were treated with 0.05mg/mL 31), freestanding PSF-based bioelectronic scaffolds similar to the PDL solution at room temperature overnight, then washed with PBS for 2–3 × 5 2 proof-of-concept shown in Fig. 2G represent a logical route to- times. Cells (passage 16) were plated at 1.6 10 cells per cm and the hiNSC maintaining medium (knockout Dulbecco’s Modified Eagle’s Medium sup- ward stable bioelectronics-embedded hybrids. plemented with 20% knockout xeno-free SR, 1% Glutamax, 1% antibiotic- As with all bioactive materials, the mechanical properties of antimycotic and 0.1 mM β-mercapthanol) was changed once every 3 d. PSF are an important consideration. A similar formulation of Immunohistochemistry. Cells were fixed with 4% paraformaldehyde at 4 °C for PSF had a reported elastic modulus of 15.6 ± 1.1 GPa (32). This 15 min and then incubated with a blocking solution consisting of 6% goat was comparable to SU8, which had elastic modulus between serum and 0.1% TritonX-100 in PBS. The primary antibody (Rabbit TUJ-1)

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.2003696117 Ju et al. Downloaded by guest on September 28, 2021 was added to the blocking buffer and incubated for 2 h at RT and then the Statistics. Calculations were performed in GraphPad Prism or OriginLab. All of sample was washed with PBS thoroughly. Cells were next incubated with the the values are represented as means ± SEM. Statistical analyses were per- fluorescent secondary antibody (Abcam anti-rabbit IgG conjugated to Alexa formed using one-way ANOVA, with P ≤ 0.05 considered statistically 488) in blocking buffer for 1 h and washed again with PBS. Nuclei were significant. counterstained by incubating cells with DAPI solution for 10 min (34). Data Availability Statement. Raw data associated with Figs. 1–5 are available . Chip fabrication and assembly details are found in SI in the Harvard/Tufts University Dataverse at https://dataverse.harvard.edu/ Appendix. Extracellular signals were measured using a 16-channel data ac- dataset.xhtml?persistentId=doi:10.7910/DVN/SXSI8O (57). quisition system (USB-ME16-FAI-System, Multichannel Systems) at a sampling Additional methods relating to silk fibroin refinement and characteriza- frequency of 25 KHz. All signals were preamplified 1,000×. They were fil- tion, chip assembly, and PSF stability, as well as SI Appendix, Figs. S1–S12,can tered with a digital bandpass filter: 1 Hz to 2.25 kHz for HL-1 cell mea- be found in SI Appendix. surements and 1 Hz to 1.25 kHz for brain slice measurements.

ACKNOWLEDGMENTS. We thank Prof. David Kaplan for invaluable advice. Mouse Heart and Brain Signal Recording. BALB/c mice (6 wk, female) were We thank Dr. Will Collins, Dr. Tao Yang, and Dr. Chengchen Guo for killed by carbon dioxide asphyxiation. Their hearts and brains were carefully experimental assistance. We acknowledge a Tufts Collaborates Grant to isolated from the surrounding tissue, then thoroughly washed with PBS (RT) B.P.T., a Tufts Research Advancement Fund award to B.P.T., a National Science to remove the blood. They were immediately interfaced with MEA devices to Foundation of China Grant (2190050415) to J.J., and a China Research Council record signals. All experiments were performed in accordance with US Animal Award ([2016]3100) to H.L. All authors thank the NIH for grant support Welfare Act and institutional guidelines and were approved by the In- through the Tissue Engineering Resource Center (P41 EB002520). This work stitutional Animal Care and Use Committee at Tufts University. was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Coordinated Infrastructure Network, which is Surface Wettability Characterization. Contact angles were evaluated using a supported by the NSF under Award 1541959. CNS is part of Harvard University. custom-built measurement apparatus and calculated with OCA 20 soft- This work was also performed in part at the Tufts Micro and Nano Fabrication ware (DataPhysics). Each surface was measured at five different positions. Facility with the assistance of Dr. James Vlahakis.

1. A. Zhang, C. M. Lieber, Nano-bioelectronics. Chem. Rev. 116, 215–257 (2016). 25. A. N. Mitropoulos et al., Transparent, nanostructured silk fibroin hydrogels with 2. H. Liu et al., : 1D nanomaterial building blocks for cellular inter- tunable mechanical properties. ACS Biomater. Sci. Eng. 1, 964–970 (2015). faces and hybrid tissues. Nano Res. 11, 5372–5399 (2018). 26. Z. Zhu et al., High-strength, durable all-silk fibroin hydrogels with versatile process- 3. Z. Luo, D. E. Weiss, Q. Liu, B. Tian, Biomimetic approaches toward smart bio-hybrid ability toward multifunctional applications. Adv. Funct. Mater. 28, 1704757 (2018). systems. Nano Res. 11, 3009–3030 (2018). 27. S. Kapoor, S. C. Kundu, Silk protein-based hydrogels: Promising advanced materials 4. M. E. Spira, A. Hai, Multi-electrode array technologies for neuroscience and cardiol- for biomedical applications. Acta Biomater. 31,17–32 (2016). ENGINEERING ogy. Nat. Nanotechnol. 8,83–94 (2013). 28. M. A. Brenckle et al., Modulated degradation of transient electronic devices through 5. O. A. Bolonduro, B. M. Duffy, A. A. Rao, L. D. Black, B. P. Timko, From biomimicry to multilayer silk fibroin pockets. ACS Appl. Mater. Interfaces 7, 19870–19875 (2015). bioelectronics: Smart materials for cardiac tissue engineering. Nano Res. 13, 29. A. R. Murphy, D. L. Kaplan, Biomedical applications of chemically-modified silk fi- 1253–1267 (2020). broin. J. Mater. Chem. 19, 6443–6450 (2009). 6. R. Feiner et al., Engineered hybrid cardiac patches with multifunctional electronics for 30. R. D. Abbott, E. P. Kimmerling, D. M. Cairns, D. L. Kaplan, Silk as a biomaterial to online monitoring and regulation of tissue function. Nat. Mater. 15, 679–685 (2016). support long-term three-dimensional tissue cultures. ACS Appl. Mater. Interfaces 8, 7. W. Ding, A. C. Wang, C. Wu, H. Gao, Z. Wang, Human-machine interfacing enabled by 21861–21868 (2016). triboelectric nanogenerators and tribotronics. Adv. Mater. Technol. 4, 1800487 (2019). 31. S. Wang et al., In vitro 3D corneal tissue model with epithelium, stroma, and in- 8. S. K. Rastogi et al., Graphene microelectrode arrays for electrical and optical mea- nervation. Biomaterials 112,1–9 (2017). surements of human -derived cardiomyocytes. Cell. Mol. Bioeng. 11, 407–418 32. N. E. Kurland, T. Dey, S. C. Kundu, V. K. Yadavalli, Precise patterning of silk micro- (2018). structures using photolithography. Adv. Mater. 25, 6207–6212 (2013). 9. B. Tian et al., Three-dimensional, flexible nanoscale field-effect transistors as localized 33. R. K. Pal et al., Fabrication of precise shape-defined particles of silk using bioprobes. Science 329, 830–834 (2010). photolithography. Eur. Polym. J. 85, 421–430 (2016). 10. A. F. McGuire, F. Santoro, B. Cui, Interfacing cells with vertical nanoscale devices: 34. D. M. Cairns et al., Expandable and rapidly differentiating human induced neural Applications and characterization. Annu. Rev. Anal. Chem. (Palo Alto, Calif.) 11, stem cell lines for multiple tissue engineering applications. Stem Cell Reports 7, 101–126 (2018). 557–570 (2016). 11. H. Liu et al., Heart-on-a-chip model with integrated extra- and intra-cellular bio- 35. D. N. Rockwood et al., Materials fabrication from Bombyx mori silk fibroin. Nat. electronics for monitoring cardiac electrophysiology under acute hypoxia. Nano Lett. Protoc. 6, 1612–1631 (2011). 20, 2585–2593 (2020). 36. S. Kim et al., All-water-based electron-beam lithography using silk as a resist. Nat. 12. B. P. Timko et al., Electrical recording from hearts with flexible nanowire device ar- Nanotechnol. 9, 306–310 (2014). rays. Nano Lett. 9, 914–918 (2009). 37. I. Mironi-Harpaz, D. Y. Wang, S. Venkatraman, D. Seliktar, Photopolymerization of 13. D.-H. Kim et al., Dissolvable films of silk fibroin for ultrathin conformal bio-integrated cell-encapsulating hydrogels: Crosslinking efficiency versus cytotoxicity. Acta Bio- electronics. Nat. Mater. 9 , 511–517 (2010). mater. 8, 1838–1848 (2012). 14. Y. Liu, M. Pharr, G. A. Salvatore, Lab-on-skin: A review of flexible and stretchable 38. C. G. Williams, A. N. Malik, T. K. Kim, P. N. Manson, J. H. Elisseeff, Variable cyto- electronics for wearable health monitoring. ACS Nano 11, 9614–9635 (2017). compatibility of six cell lines with photoinitiators used for polymerizing hydrogels and 15. X. Dai, G. Hong, T. Gao, C. M. Lieber, Mesh nanoelectronics: Seamless integration of cell encapsulation. Biomaterials 26, 1211–1218 (2005). electronics with tissues. Acc. Chem. Res. 51, 309–318 (2018). 39. C. Xie et al., Three-dimensional macroporous nanoelectronic networks as minimally 16. X. Dai, W. Zhou, T. Gao, J. Liu, C. M. Lieber, Three-dimensional mapping and regu- invasive brain probes. Nat. Mater. 14, 1286–1292 (2015). lation of action potential propagation in nanoelectronics-innervated tissues. Nat. 40. W. C. Claycomb et al., HL-1 cells: A cardiac muscle cell line that contracts and retains Nanotechnol. 11, 776–782 (2016). phenotypic characteristics of the adult cardiomyocyte. Proc. Natl. Acad. Sci. U.S.A. 95, 17. A. Kalmykov et al., Organ-on-e-chip: Three-dimensional self-rolled array for 2979–2984 (1998). electrical interrogations of humans electrogenic spheroids. Sci. Adv. 5, eaax0729 41. V. N. Vernekar et al., SU-8 2000 rendered cytocompatible for neuronal bioMEMS (2019). applications. J. Biomed. Mater. Res. A 89, 138–151 (2009). 18. Q. Li et al., Cyborg organoids: Implantation of nanoelectronics via organogenesis for 42. Y. Zhang et al, “Application of SU-8 as the insulator toward a novel planar micro- tissue-wide electrophysiology. Nano Lett. 19, 5781–5789 (2019). electrode array for extracellular neural recording,” in 2010 IEEE 5th International 19. T.-M. Fu et al., Stable long-term chronic brain mapping at the single-neuron level. Conference on Nano/Micro Engineered and Molecular Systems (Institute of Electrical Nat. Methods 13, 875–882 (2016). and Electronics Engineers, Piscataway, NJ, 2010), pp. 395-398. 20. G. Hong et al., A method for single-neuron chronic recording from the retina in 43. F. H. Gage, S. Temple, Neural stem cells: Generating and regenerating the brain. awake mice. Science 360, 1447–1451 (2018). Neuron 80, 588–601 (2013). 21. W. S. W. Harun et al., A comprehensive review of hydroxyapatite-based coatings 44. E. Moy, F. Y. L. H. Lin, J. W. Vogtle, Z. Policova, A. W. Neumann, Contact angle studies adhesion on metallic biomaterials. Ceram. Int. 44, 1250–1268 (2018). of the surface properties of covalently bonded poly-L-lysine to surfaces treated by 22. V. Miguez-Pacheco, L. L. Hench, A. R. Boccaccini, Bioactive glasses beyond bone and glow-discharge. Colloid Polym. Sci. 272, 1245–1251 (1994). teeth: Emerging applications in contact with soft tissues. Acta Biomater. 13,1–15 45. B. Valamehr et al., Hydrophobic surfaces for enhanced differentiation of embryonic (2015). stem cell-derived embryoid bodies. Proc. Natl. Acad. Sci. U.S.A. 105, 14459–14464 23. A. J. Salinas, M. Vallet-Regí, Bioactive ceramics: From bone grafts to tissue engi- (2008). neering. RSC Adv. 3, 11116–11131 (2013). 46. Y. H. Kim, N. S. Baek, Y. H. Han, M.-A. Chung, S.-D. Jung, Enhancement of neuronal 24. A. Khademhosseini, R. Langer, A decade of progress in tissue engineering. Nat. Pro- cell adhesion by covalent binding of poly-D-lysine. J. Neurosci. Methods 202,38–44 toc. 11, 1775–1781 (2016). (2011).

Ju et al. PNAS Latest Articles | 7of8 Downloaded by guest on September 28, 2021 47. A. S. Lammel, X. Hu, S.-H. Park, D. L. Kaplan, T. R. Scheibel, Controlling silk fibroin 52. K. Min, M. Umar, S. Ryu, S. Lee, S. Kim, Silk protein as a new optically transparent particle features for drug delivery. Biomaterials 31, 4583–4591 (2010). adhesion layer for an ultra-smooth sub-10 nm gold layer. Nanotechnology 28, 115201 48. A. Blau, Cell adhesion promotion strategies for signal transduction enhancement in (2017). microelectrode array in vitro electrophysiology: An introductory overview and critical 53. J. Chen, H. Venkatesan, J. Hu, Chemically modified silk proteins. Adv. Eng. Mater. 20, 1700961 (2018). discussion. Curr. Opin. Colloid Interface Sci. 18, 481–492 (2013). 54. S. H. Kim et al., Precisely printable and biocompatible silk fibroin bioink for digital 49. C. J. Robin, A. Vishnoi, K. N. Jonnalagadda, Mechanical behavior and anisotropy of light processing 3D printing. Nat. Commun. 9, 1620 (2018). spin-coated SU-8 thin films for MEMS. J. Microelectromech. Syst. 23, 168–180 55. M. G. Mohammed, R. Kramer, All-printed flexible and stretchable electronics. Adv. (2014). Mater. 29, 1604965 (2017). 50. C. Jiang et al., Mechanical properties of robust ultrathin silk fibroin films. Adv. Funct. 56. L. Grob et al., Printed 3D electrode arrays with micrometer‐scale lateral resolution for – Mater. 17, 2229 2237 (2007). extracellular recording of action potentials. Adv. Mater. Technol. 5, 1900517 (2019). 51. R. Feng, R. J. Farris, Influence of processing conditions on the thermal and mechanical 57. J. Ju, N. Hu, D. M. Cairns, H. Liu, B. P. Timko, “Photo-crosslinkable, insulating silk fi- properties of SU8 negative photoresist coatings. J. Micromech. Microeng. 13,80–88 broin for bioelectronics with enhanced cell affinity.” Harvard/Tufts University Data- (2003). verse. https://doi.org/10.7910/DVN/SXSI8O. Deposited 11 May 2020.

8of8 | www.pnas.org/cgi/doi/10.1073/pnas.2003696117 Ju et al. Downloaded by guest on September 28, 2021