Bacterial Cellulose as a Supersoft Neural Interfacing Substrate - ACS

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Biological and Medical Applications of Materials and Interfaces

Bacterial Cellulose as a Supersoft Neural Interfacing Substrate Junchuan Yang, Mingde Du, Le Wang, Sixiang LI, Guorui Wang, Xinglong Yang, Lijuan Zhang, Ying Fang, Wenfu Zheng, Guang Yang, and Xingyu Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b12083 • Publication Date (Web): 12 Sep 2018 Downloaded from http://pubs.acs.org on September 13, 2018

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ACS Applied Materials & Interfaces

Bacterial Cellulose as a Supersoft Neural Interfacing Substrate Junchuan Yang,†,‡,⊥ Mingde Du,†,§,⊥ Le Wang,†,⊥ Sixiang Li,‡ Guorui Wang,† Xinglong Yang,†,§ Lijuan Zhang,† Ying Fang,† Wenfu Zheng,†,* Guang Yang,‡,* Xingyu Jiang†,§,* †

Beijing Engineering Research Center for BioNanotechnology, CAS Key Lab for

Biological Effects of Nanomaterials and Nanosafety, CAS Center of Excellence for Nanoscience, National Center for NanoScience and Technology, Beijing, 100190, China. ‡

National Engineering Research Center for Nano-Medicine, Department of

Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China. §

University of Chinese Academy of Sciences, Beijing, 100049, China.

*E-mail: [email protected] (X. Jiang); [email protected] (G. Yang); [email protected] (W. Zheng). ⊥

Junchuan Yang, Mingde Du and Le Wang contributed equally to this work.

ABSTRACT: Biocompatible neural interfaces hold great promise for treating neurological disorders and enhancing the mental and physical ability of human beings. Most of the currently available neural interfaces are made from rigid, dense inorganic materials that cause tissue damage. We present supersoft multichannel electrodes by depositing gold layers on thin bacterial cellulose (BC) (Au-BC electrodes). The Young’s modulus of BC (EBC=120 kPa) is between those of the brain tissue (Ebrain=2.7~3.1 kPa) and the peripheral neural tissues (Eperipheral nerve=580~840 kPa). The bending stiffness of the Au-BC electrodes corresponds to 1/5200 of Au-polyimide electrodes with the same layout. Furthermore, the Au-BC electrodes is highly durable (conductivity >95% after 100 cycles of 180o bending). In vivo recording of brain 1

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electric activity demonstrates the great potential of the Au-BC electrodes for neural interfacing applications.

Keywords: electrode array, conformable, soft electrodes, bacterial cellulose, neural interface.

1. INTRODUCTION Flexible electrodes hold great promise for treating neurological disorders or enhancing the mental and physical ability of human beings. However the mismatch between the rigid and planar surfaces of electrodes (usually silicon-based insulating substrate with metallic interconnects) and the soft and three-dimensional (3D) features of native neural tissues can cause unwanted biological responses and pose significant challenges in neural surface interfacing.1-3 The factors affecting the contact of the electrodes with the tissues are mainly associated with the bending stiffness of the electrode substrates.4 A reduction in the substrate thickness and a decrease in the Young’s modulus of the substrates are effective strategies to reduce the bending stiffness.1 Thus softer and thinner electrodes are highly desirable for neural surface interfacing.5 Recently, researchers have developed soft electrode materials6-9 and metal electrodes based on soft substrates such as polyimide (PI),1, 10-13 parylene C,14-16 polycarbonate (PC),17-19 polydimethylsiloxane (PDMS) 20-24 and shape memory polymers (SMP).25-26 As compared with silicon-based electrodes, these soft electrodes induce less neural tissue damage and allow stable neural communication.27 However, these synthetic polymers are still much more rigid (in the range of Epolymer substrates=1.00×10

3

kPa to 8.45×106 kPa) than soft neural tissues (Ebrain=2.7~3.1 kPa,

Eperipheral nerve=580~840 kPa),28 which prevents the electrodes from fully conforming to neural tissues (Table S1). Thus, electrodes that have mechanical properties more closely matching native neural tissues are highly desirable, though difficult to fabricate. 2


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As natural materials, celluloses have become attractive candidates for paper based devices due to their high flexibility, biocompatibility, and low cost.29-30 As a kind of natural nanocellulose, bacterial cellulose (BC) is a natural polysaccharide (C6H10O5)n (Figure S1). The hydrated BC has a porous structure containing a large percentage of water (99%), which resembles the extracellular matrix of tissues. Thus, BC is an excellent material to serve as matrix for tissue engineering. BC has been widely used in biomedical engineering,31-32 and functional electric devices 33-36 due to its outstanding properties in terms of super softness, surface porosity, optical transparency, and biocompatibility.37-38 Although there are reports on paper-based electrodes29-30, 39 and conductive cellulose composites,35, 40-41 unfortunately, the single electrode of reported paper-based electronics is of several hundreds of micrometers in width, which is too large to be used as a neural electrode. There is not yet a method to build multi-channel micro neural electrodes on BC. BC is a hydrogel which cannot be processed by current micro-fabrication strategies such as photolithography42-43 or chemical etching. As a result, the utilization of BC as a substrate to build high-throughput microelectronic neural interfaces has not been reported. In this study, we present a novel strategy to fabricate microscale thin film Au electrodes based on BC substrates. The natural BC-based electrodes have advantages over the electrodes based on synthetic polymers: i) superior compliance, with a Young’s modulus (EBC=80~120 kPa) somewhere in between those of the peripheral neural tissues (Eperipheral nerve=580~840 kPa) and the brain tissues (Ebrain=2.7~3.1 kPa) (Figure 1, Table S1),12 and a bending stiffness that is 1/5200 of a PI-based Au electrode; ii) the thickness of the electrodes and the width of a single channel can be as thin as 5 µm, which are smaller than a single mammalian cell; iii) extreme flexibility and robustness, with a conductivity decrease of less than 5% after 100 cycles of 180o bending; iv) high electrode surface roughness (Rq) (15.6 times larger than that of PI-based Au electrodes), which can help promote conformal contact with the curvilinear surfaces of the neural tissues; v) high biocompatibility, wet BC is actually a hydrogel which 3



resembles extracellular matrix; and vi) straightforward and environmentally friendly fabrication procedure, we directly deposit gold circuit patterns on the BC surface with a shadow mask.1, 10 Long-term subcutaneous implantation and neural recordings in rats demonstrate the high safety and neural interfacing potential of Au-BC electrodes.

2. MATERIALS AND METHODS 2.1 Manufacture of Silicon (Si) Shadow Mask. Reactive ion etching (RIE) was used to fabricate a thin (200 µm) silicon shadow mask (Figure S2). Double-sided polished Si wafers with a thickness of 200 µm were used for this experiment. For the first step, a layer of 7 µm positive photoresist SUN 1150p (Suntific, China) was spin-coated (2000 rpm, 30 s) onto the top side of the Si wafer. The Si wafer was sequentially treated at 100 °C for 5 min, exposed to UV (10 mJ·cm–2·s–1, 30 s), treated in developer solution for 3 min to remove the uncured photoresist and hard baked at 100 oC for 5 min. We coated the bottom side of the Si wafer with a layer of 3 µm positive photoresist using the same method. The second step was RIE etching by Inductively Coupled Plasma Etching System for Silicon and Silicon Dioxide (Plasmalab System 100 ICP180, UK). A layer of 175 µm naked Si was etched using RIE from the top side and sequentially a layer of 25 µm of naked Si was etched using the same method from the bottom side. We used acetone to remove the photoresist and harvest the hollow Si shadow mask. The area coated with photoresist was protected from etching, while the area without photoresist was etched. The hollow Si template was used as shadow mask in the following experiments. The width of the Au electrodes fabricated using this method was available from 5 µm to 100 µm. In order to precisely record CNS signal, we need electrodes with a small size as the neural cells have an average size of 20 µm. So, we built 20 µm wide microelectrode channels on the BC substrate. 2.2 Manufacture of Ultra-Thin BC Substrate. The BC was produced by fermentation of Acetobacter xylinum (ATCC53582) in Hestrin and Schramm (HS) 4


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medium at 30 oC. The analytical pure reagents for produce of BC were purchased from Sinopharm Chemical Reagent Co., Ltd. The HS medium was composed of 20 g L-1 glucose, 5 g L-1 yeast extract, 5 g L-1 peptone, 6.8 g L-1 disodium hydrogen phosphate dodecahydrate, and 1.5 g L-1 citric acid monohydrate. BC is a metabolic by-product of bacterial fermentation, meaning that the thickness is increased during increased culture times. The adjustable thickness of the BC film in lab research ranges from micrometers to centimeters. After culture, we harvested the BC and purified it in 1.0 % NaOH solution at 100 oC for 1 h (twice), 1.5 % acetic acid for 30 min and subsequently in deionized water at RT until neutral pH was reestablished. The purified BC was autoclaved (121 oC, 1.1 bar for 30 min) and stored in deionized water at 4 oC. We prepared the ultra-thin BC film by hot-pressing raw BC film for 12-24 h (100 o

C, 10 MPa) to remove the absorbed water with a hot-press machine, until the BC

films became totally dry and smooth. We can control the thickness of the raw BC by tuning the cultivation time, and produce hot-pressed BC with different final thickness by selecting raw BC of different thicknesses and different hot-pressing parameters (Table S2). 2.3 Manufacture of Electrode arrays. The 10-channel electrode arrays were fabricated as follows (Figure 2a): we prepared the ultra-thin BC films by hot-pressing raw BC films. We spread the thin BC film onto a Si slide and kept it totally adhering to the surface of Si slide. We mounted the mask1 onto the BC surface. A pattern of Cr/Au (5/200 nm) layer was deposited through the shadow mask1 onto the BC substrate by electron beam evaporation. Then we removed the shadow mask1, and mounted shadow mask2 (Figure S3), subsequently, a Si3N4 insulating layer (800 nm) grew onto the Au wires by low temperature plasma-enhanced chemical vapor deposition (LT-PECVD). We mounted the shadow mask2 on the Au pattern by careful alignment using a stereo microscope. Since the diameter of the holes on the shadow mask2 was larger than the diameter of Au wires, the whole Au wires except the working area were fully coated by Si3N4 by careful 5



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alignment. A 20 × 20 µm naked Au area was formed at the end of each channel of 10-channel recording electrodes. The recording electrodes were designed to be used for electrocorticography, which involves recording electrical activity from the brain’s surface. The anisotropic conductive adhesive tape (MF-331, Hitachi, Japan) was inserted between the Au contact pads on the BC and the flexible flat cable (FFC) (Figure 2b). The high throughout FFC was fabricated by industrial screen-printing tin wires on 200 µm thick PI. The contact pads arrays on the BC were carefully aligned with the tin wires on FFC. The sandwich structured devices were clamped and heated using a hot-pressing machine at 120 oC for 1 min. This process generated a strong mechanical bond between the electrode arrays on BC and the FFC with low contact resistance. The FFC was used to connect the electrodes with the neural signal recording equipment. 2.4 Morphology Characterization. . We observed the surface morphology and the cross sectional morphology of electrodes by scanning electron microscope (SEM, S4800, Japan). The sample for natural raw BC hydrogel was prepared by freeze-dry treatment. We utilized atomic force microscopy (AFM, Dimension Icon, Veeco) in the standard tapping mode to measure the surface roughness of Au-BC, BC, Au-PI, and PI. Scanning area: 20 µm × 20 µm. Each group consists of five samples. We reported the average value with standard deviation. 2.5 Hydrophilicity. We evaluated the wettability of hot-pressed BC with water contact angle analysis system (DSA100, Germany), setting PI (2611, DUPONT, USA), parylene C, PC and PDMS as the controls. Deionized water (MiliQ deionizer, Millipore) with a droplet size of 3 µL was used for all contact angle measurements. 2.6 Light Transmittance. We analyzed the absorbance value (A) of light through BC or PI films with wavelength ranging from 380 nm to 780 nm by UV-vis spectrophotometry (UV2450, Shimadzu, Japan), the dry hot-pressed BC, PI, and rehydrated hot-pressed BC (rehydrated BC) films were measured respectively. The transmittance ratio (T) was analyzed using the following equation: 6


.

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2.7 Thermogravimetric Analysis (TGA). We carried out TGA measurements for the BC and Au-BC films with a thermogravimetric analyzer Model Pyris1 TGA (Perkin Elmer Instruments, China). The degradation of the films was investigated from 25 to 600 oC, under nitrogen atmosphere, at a heating rate of 10 oC min-1. 2.8 Mechanical Property Analysis. We measured the tensile strength of the dry hot-pressed BC, rehydrated hot-pressed BC, wet raw BC with a universal mechanical analyzer (Instron5567, USA). We cut the samples into dumbbell-shaped specimens and set the distance between the two clamps as 50 mm. All the samples were tested at a stretching speed of 20 mm min-1 at 25 oC, 50 % relative humidity. As the insulation layer can be constructed by SU-8, PI or Si3N4 using the same method with the same parameters, we neglected the insulation layer and mainly analyzed the total bending stiffness of Au-BC electrodes and Au-PI electrodes as a function of the thickness of the supporting substrates. The cross sectional geometry of the electrode arrays was illustrated in Figure S4. There are n gold electrodes (width bm=20 µm, height hm=200 nm, Young’s modulus EAu=78 GPa) surrounded by substrate (width b, height h, h′ is the distance between bottoms of the gold electrodes and the thin film. Young’s modulus EPI=2.5 GPa, Edry hot-pressed BC=3.9 MPa, Ewet hot-pressed BC=0.12 MPa). We calculate EI as h′ increases from 0 to 45 µm by 0.5 µm increment using the following equation. 1 The distance between the neutral axis and bottom of the thin film is

(1) The bending stiffness of the thin film is

(2) 2.9 Stability Test. To test the flexibility of Au-BC electrodes, the wet device was 7



repeatedly bent 180o for 100, 200 and 300 times with a custom-built bending apparatus, and the electrical conductivity and electrical resistivity were respectively measured using a standard four point-probe system with a Keithley 2400 current source and an HP 34420A nanovoltmeter. Each group consisted of five samples. At least five specimens were tested for each sample, we reported the average value with standard deviation. To test the stability of Au-BC electrodes, we incubated the Au-BC electrodes in cerebrospinal fluid at 37 oC mimicking in vivo environment, and tested the degeneration ratio, electrical resistivity, and mechanical property at day 0, day 14 and day 28. The degeneration ratios of the electrodes were performed by measuring the mass loss at different time points. The electrical resistivity of the electrode was measured by methods described in Section 2.11. The mechanical property measurement of the electrodes was measured by methods described in Section 2.8. To evaluate the stability of the electrodes, the Au/BC interface after 28 days of incubation was imaged by SEM. 2.10 Electrochemical Characterization. We investigated the interfacial impedance of the fabricated electrodes in an artificial cerebrospinal fluid using electrochemical impedance spectroscopy. We conducted the electrochemical experiments of the electrodes at room temperature using an Autolab system (PGSTAT 302N, NOVA software, Ecochemie, Utrecht, The Netherlands). We used the conventional three electrodes system to determine the impedance. The system included fabricated electrode as a working electrode, a Pt wire as a counter electrode, and Ag/AgCl as a reference electrode. We measured the impedance between the electrode and the electrolyte over a frequency range of 1~105 Hz. 2.11 Biosafety Evaluation. We evaluated the cytotoxicity of the PC-12 neural cells on the electrodes surface by live/dead staining. We cultured the cells with DMEM/F12 (Dulbecco's Modified Eagle Media: Nutrient Mixture F-12, Gibco) containing 10% HBS (horse bovine serum, Gibco), 1% PS (penicillin-streptomycin, Gibco) at 37 °C, 8


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5% CO2. The subculture of the cells was conducted by digestion with 0.25% trypsin every 3 days. In this study, both the BC and Au-BC samples were sterilized at 121°C for 30 min by autoclave before cell culture. The cells were seeded on the surface of the electrodes with a density of 104~105 mL-1 and incubated for 3 days. Then we stained the cells with the dual fluorescence Calcein AM/PI (Dojindo, Japan) and observed the cells with a laser confocal scanning microscopy (Zeiss LCSM 710). We also observed the cell morphology on Au-BC electrodes by staining cell nuclei and the F-actin filaments with Hoechst 33342 (Sigma) and Alexa Fluor 568 phalloidin (Invitrogen), respectively. We measured the hemolysis activity of the material with fresh human blood. We collected the erythrocytes by centrifuging for 3 times (1500 rpm, 15 min) in 4 oC saline solution, then resuspended the erythrocytes in saline solution to prepare 2% w/v solution44. For the experiment group, we cut the Au-BC and PI-BC into small round shaped films and put them into the bottom of a 24 well plate. We set saline as negative control and deionized water as positive control. We added 500 µL blood solution into each well of the plate. After incubation for 3 h at 37 oC, 100 µL solution of each sample was transferred into a new 96 well plate. We measured the hemoglobin release spectrophotometrically by UV-Vis analysis at 540 nm absorbance after centrifugation at 1500 rpm for 15 min. Each group included five samples. We reported the average value with standard deviation. 2.12 Animal Experiment. In this study, Sprague-Dawley male rats (250 g, n=3) were anaesthetized with 20 % urethane (5 mL kg-1) and fixed in a stereotaxic frame. A 4 mm × 4 mm craniotomy was performed between the bregma and lambda of the skull and the electrodes were put over the left cortical surface of the rat brain to record the electrocorticography (ECoG) activity (Figure 2c). We record the ECoG signal of the brain with the Au-BC electrodes using a multichannel acquisition system (Cerebus; Blackrock microsystems, USA). Channels 1~6 marked in Figure S3 were used to record the signals. The flexible electrodes were closely adhered to the brain surface 9



due to its ultrasoftness and hydrophilic surface. Acute recording was performed at 40 kHz sampling rate with 50-Hz notch filter to remove interference from electrical power line. NeuroExplorer program (Nex Technologies, Madison, AL, USA) was used for data analysis. We intraperitoneally injected the rat with penicillin-G sodium (8 mU kg-1) to induce epileptic activities. We performed all the animal experiments according to the guideline of the Institutional Animal Care and Use Committee of the National Center for Nanoscience and Technology of China. 2.13 Histological Analysis. We implanted the electrodes into cerebral cortex to evaluate their long-term biocompatibility. In this study, male C57 mice (n=10) were anesthetized with pelltobarbitalum natricum (100 mg kg-1) and fixed on in a stereotaxic frame. The brain was exposed after craniotomy, and the Au-PI electrodes and Au-BC electrodes were put over the left and right cortical surface respectively. The craniotomy was filled with Kwik-Sil silicone elastomer (World Precision Instruments, Inc). The skin was sutured, and the dental cement was coated on the incision after suture. The mice were sacrificed at 2 weeks (n=5) and 4 weeks (n=5) and taken for immunohistological analysis to evaluate the biocompatibility. We anesthetized the mice by intraperitoneal injection of pelltobarbitalum natricum (100 mg kg-1) and then perfused the mice transcardially with 4% paraformaldehyde in PBS. After removing the skin and skull, the brain was fixed with 4% formaldehyde and sectioned to 100 µm thick slices. The slices were stained with three antibodies, NeuN (neuronal nuclei marker), GFAP (glial marker), and DAPI (nuclei marker) and observed with a laser confocal microscopy. We analyze the intensity of astrocytes reaction by measuring the relative fluorescence intensity of astrocytes in Au-BC and Au-PI at 2 and 4 weeks using Image J software. The fluorescence intensity was got by measuring the brightness of pixel areas from the electrode-brain interface to the tissues 50 µm beneath the interface. The Au-BC electrodes and Au-PI electrodes with the same thickness of 20 µm were subcutaneously implanted onto the back of rats (SD, female, 250 g, SPF grade, 10


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HFK Bioscience Co., Ltd, Beijing) to evaluate their long-term (8 weeks) biocompatibility (n=40). The rats were randomly divided into two groups. The Au-BC electrodes and Au-PI electrodes, with same thickness, size (0.6 mm × 0.6 mm) and same Au circuits on their surface, were used to evaluate the biocompatibility. The rats were anesthetized with 3% (wt vol-1) pentobarbital sodium (Sigma, 1 mL 100 g-1) and implanted with Au-BC and Au-PI electrodes respectively at the left and right back, sequentially sutured after the surgery. 5 rats were sacrificed at 1, 2, 4 and 8 weeks respectively, and the implants and surrounding tissues were dissected to take hematoxylin and eosin (HE) staining and observed by an inverted microscope (Leica DM 4000M, Germany).

3. RESULTS AND DISCUSSION 3.1 Morphological Characterization of Au-BC Electrodes. The main challenge in developing ultra-thin Au-BC electrode arrays is the mesoporous structure and uneven surface of raw BC films, the surface roughness which greatly hinders the physical connectivity of the microelectrode array makes it challenging to create thin film electronic structures. So far no strategy to build microelectrode arrays on BC has been reported. We however think that the hot-pressing of the raw BC hydrogel might allow both dehydration of the material and the smoothening of the surface to allow the BC to be compatible with microfabrication. Such dehydrated BC can accommodate the deposition of metallic materials such as Au via electron-beam evaporation in high vacuum, without compromising its mechanical, chemical, and biological properties. We produce BC by fermentation of Acetobacter xylinum, and the thickness of the BC increases with the increasing culture time (Figure S5). We use SEM to examine the cross-section of the hot-pressed BC films with different thicknesses ranging from 5 µm to 100 µm (Figure 3a). Considering 5 µm substrates are too thin and soft to handle, we use 10 µm substrates in the following experiments. 11



We examine morphological variations of BC films after the hot-pressing treatment. The raw BC films have a highly porous structure in macroscale which can lead to the breakage of the conductive Au layer (Figure 3b). After the hot-pressing treatment the surface becomes compact (Figure 3c). After deposition of a 200 nm-thick Au layer, the gaps and pores between adjacent nanofibers are filled with Au nanoparticles, and the surface of the Au-BC is smoother than of the hot-pressed BC (Figure 3d). A layer of 200 nm Au circuits is projected onto a thin BC substrate with a thickness of 10 µm (Figure 3e). We study the range of the widths of Au ribbon that can be fabricated using this method. The Au ribbons with width ranging from 5 to 100 µm are projected onto the surface of BC, proving that this method is robust enough to build microelectrodes with a width down to 5 µm (Figure 3f). We fabricate 10-channel electrodes with 20 µm working electrodes for the following experiments (Figure 3g~ i), utilizing the corresponding shadow masks. AFM analysis indicates that the root-mean-squared roughness of Au-BC is 15.6 times larger than that of Au-PI (3.4 nm) (Figure 3j), which means the hot-pressing method is effective in reducing the possible breakage of conductive Au layer caused by the porous structure in macroscale, but still keep its roughness of the nanofiber in nanoscale. Due to the rough property of neural tissue surface, the Au-BC can maintain more contact surface with the curvilinear surfaces of neural tissues than the Au-PI. 3.2 Thermal Stability Analysis of Au-BC Electrodes. We investigate the thermal stability of the hot-pressed BC and Au-BC films by TGA, because the manufacturing processes for Au-BC electrodes involves high-temperature treatments ranging from 150 oC to 200 oC such as hot-pressing of the raw BC, electron beam evaporation of the Au layer, and chemical vapor deposition of Si3N4. The elevation of the temperature to 300 oC only induces slight weight loss for dry hot-pressed BC (5.1%) and dry Au-BC (6.4%) (Figure 4a). The TGA analysis confirms that the BC substrate remains intact at temperatures lower than 300 oC which is much higher than the highest temperature (200 oC) in the Au-BC manufacturing processes.45 12


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3.3 Transmittance Analysis of Au-BC Electrodes. We test the transparency of hot-pressed BC films because high transparency of electrodes is essential for locating the interfacing sites in vivo. For example, when we put the recording electrodes onto the target site of a human brain model, we can see the detailed structures of the brain tissues through the highly transparent electrodes, which can further help us observe the morphological change of the tissues beneath the electrodes (Figure S6). The rehydrated BC film displays a higher light transmission than the PI film from 380 to 550 nm, the light transmission of either group is similar from 550 to 780 nm (Figure 4b). The light transmission of the dry hot-pressed BC films is relatively low compared with the wet BC film. 3.4 Mechanical and Electrochemical Properties of Au-BC Electrodes. We investigate the electrochemical properties of the Au-BC and Au-PI electrodes by comparing their electrochemical impedance. The impedance of the Au-PI electrodes is 70.1 ± 5.8 kΩ at 1 kHz, the impedance of the Au-BC electrodes is 55.6 ± 5.0 kΩ at 1 kHz (Figure 4c). To verify the consistency in different channels of the Au-BC electrodes, we test the impedance of 6 channels of the Au-BC electrodes which show similar and consistent results (Figure S7a). The slight difference between the two electrodes may be ascribed to the different surface morphology of them. As shown in the AFM results (Figure 3j), the surface of Au layer on BC is rougher than that of the Au layer on PI, which means there are more contacting area between the wire and the electrode, and subsequently there will be larger electric current through the electrode and show lower impedance. The large impedance demonstrates that there is no obvious leakage through our thin Si3N4 insulating layer. We further carry out phase plot analysis, the phase angle of Au-BC (37.5o) and Au-PI (36.2o) at 1 kHz impedance is similar (Figure S7b). Thus, the electrochemical properties of two kinds of electrodes are similar. We compare mechanical properties of dry hot-pressed BC, rehydrated hot-pressed BC and PI (Figure 4d, Table S3). The elongation ratio at break of the rehydrated BC 13



reaches 17.7%, which is smaller than that of the PI (91%). However, the stress at break for the rehydrated BC is about 20 MPa, which is much lower than that of the PI (>120 MPa). The Young’s modulus of the rehydrated BC is as low as 120 kPa, which is dramatically lower than that of PI (2.5×106 kPa). Thus, although the flexibility of PI is better than that of the hydrated BC, the latter is much “softer” than the former. We further compare the bending stiffness of Au-BC and Au-PI electrodes with the same layout (Figure S4). The optimal bending stiffness of the electrodes for CNS and PNS should be near the Young’s modulus of the native brain tissues (Ebrain=2.7~3.1 kPa) and the peripheral nerve tissues (Eperipheral nerve=580~840 kPa) respectively. The Young’s modulus of BC (EBC=120 kPa) is between those of the brain tissues (Ebrain=2.7~3.1 kPa) and the peripheral nerve tissues (Eperipheral nerve=580~840 kPa), while the Young’s modulus of PI is 2.5×106 KPa, and EPI= 2×104 EBC. In this study, the bending stiffness of the electrodes increases with the increase of the substrate thickness. We calculate the data points from 0 to 45 µm by 0.5 µm increment and get 90 data points. With a thickness of 10 µm, the bending stiffness of Au-PI is 5200 times larger than that of the rehydrated Au-BC (Figure 4e). The combination of the flexibility and stiffness (Young’s modulus, bending stiffness) demonstrates that the mechanical property of BC-based electrodes is more close to the brain tissue than that of PI-based electrodes. We quantify the durability and reliability of the Au-BC by fatigue tests. Electrodes with a 200 nm Au layer on 10 µm thick BC were used to take the test. The Au-BC is repeatedly bent up and down by 180o. After bending for 0, 100, 200 and 300 cycles, the electrical resistivity of the electrodes are 5.2 ± 0.1, 5.5 ± 0.2, 5.6 ± 0.3 and 6.0 ± 0.2 mΩ·cm respectively. After repeated bending for 100, 200 and 300 cycles, the resistivity slightly increases 5.1%, 7.3% and 14.2% respectively (Figure 4f, Table S4). The transparent and supersoft Au-BC electrodes can bend and conformally attach onto cerebral sulci (Figure S6), while the rigid Au-PI electrodes cannot bend and conformally attach to the curvilinear surfaces of a human brain model (Figure S6). The Au-BC electrodes remain intact after repeated twisting and untwisting for 100 cycles, 14


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demonstrating the excellent durability and flexibility of the electrodes (Figure 5a). The SEM observation proves that both Au-PI and Au-BC (rehydrated) electrodes keep intact surface morphology after repeatedly bending for 300 times (Figure S8). The insulation layer of dry Au-BC electrodes is fragile and easy to break after repeated bending, while the insulation layer is flexible in rehydrated Au-BC electrodes. After 300 times of bending at 180o, the hydrated Si3N4 layer on the Au-BC electrodes still keeps intact surface morphology (Figure S9). We also test the stability of Au-BC electrodes by keeping them in cerebrospinal fluid (37 oC) simulating the in vivo environment. The Au-BC electrodes do not show significant degradation after 4 weeks of incubation (Figure S10a). The electrical resistivity of the electrodes does not change significantly after 2 and 4 weeks of incubation (Figure S10b), and the impedance of the Au-BC electrodes on day 0, 14, and 28 also does not show apparent change (Figure S10c). After incubation in artificial cerebrospinal fluid for 4 weeks, we also test the electrochemical impedance of electrodes before and after bending for 90o to simulate the electrode conforming to the surface of the brain, there is no significant difference when the electrode is bent or extended (Figure S10d). The elongation at break and tensile strength of BC at 4th week (17.8%, 17.9 MPa) and 2nd week (17.5%, 17.6 MPa) are similar to those on day 0 (17.1%, 17.4 MPa) (Figure S10e). Furthermore, we image the interface of the Au-BC on 4 weeks by SEM which shows the intact morphology of the Cr/Au layer, demonstrating the robustness of the Au-BC electrodes (Figure S11). The results demonstrate the high stability of the Au-BC electrodes in environment mimicking physiological condition. 3.5 Biosafety Evaluation of Au-BC Electrodes. We further evaluate the biocompatibility of the Au layer-coated BC film (Au-BC) and Au layer-coated PI film (Au-BC). We first test the biosafety of the Au-BC by hemolysis assay, which is an important biomedical parameter for assessing the safety of implantable devices. Neither the Au-BC nor the Au-PI damage human erythrocytes (Figure 5b). The live/dead staining image shows that PC-12 cells, a model neuron cell line, adhere and 15



spread well on both the BC and Au surfaces of the Au-BC (Figure 5c). 97 ± 1% cells are stained with Calcein AM, showing the good biocompatibility of the Au-BC. Cytoskeleton staining shows that PC-12 cells on both BC and Au-BC surface attach and stretch well (Figure 6b) similar with the cells cultured on petri dish (Figure 6a). The typical long protrusions (dendrites) of the cells and the orange-only staining indicate excellent biocompatibility of the Au-BC, which implies its potential for good performance in the implantation assay.46 The BC film, which is highly hydrophilic and soft as native tissues (Figure S12), is a desirable substrate material for preparing electrodes for neural interfacing. 3.6 In vivo Neural Signal Recording. Using electrodes to record the ECoG activity has been widely used in diagnosing and evaluating the status of epileptic patients. We utilize the Au-BC electrodes to record penicillin-induced epileptic activity from the randomly selected left temporal lobe of rat cerebral cortex. The typical spectra of epileptiform (5-25 Hz) generated by synchronous firing of neuronal clusters gradually increases from the latent period to epileptiform period (Figure 7a).47After the intraperitoneal injection of penicillin G sodium for several minutes, as compared with the latent ECoG signal, rhythmic epileptiform discharges are recorded by the Au-BC electrodes (Figure 7b). Penicillin can block gamma-amino butyric acid (GABA) receptors of the pyramidal neurons and lead to rhythmic epileptiform discharges (Figure 7c, d). We further record the ECoG activity of the rat brain by multichannel mapping using the Au-BC electrodes, which demonstrates the high-throughput signal readout ability of the electrodes (Figure S13). 3.7 Long-Term Implantation Evaluation. We implant the Au-BC electrodes and Au-PI electrodes into mouse cerebral cortex to evaluate their biosafety during long-term implantation. We stain the brain tissues by neuronalnucleiantigen (NeuN) and glial fibrillary acidic protein (GFAP) for staining of neurons and astrocytes respectively. The accumulation and proliferation of astrocytes in the CNS could be regarded as a form of inflammatory response. We quantify the fluorescence intensity of 16


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NeuN and GFAP using Image J software. First, the image is converted into 16-bit gray image, then the fluorescence signal of the dark area without any tissue is set as baseline, and the areas of tissues outlined by red dotted lines are chosen to calculate the fluorescence intensity (Figure S14). The level of NeuN in Au-BC and Au-PI groups decrease at 4th week compared to that at 2nd week (Figure 8a, c). Comparatively, the fluorescence of NeuN in the Au-PI group is slightly higher than that in the Au-BC group at both 2nd week and 4th week, which perhaps comes from the distortion of the tissues during imaging (Figure 8a, c). Mild inflammatory reactivity is observed in both Au-PI and Au-BC group at the implantation site in the mouse cortex 2 weeks post-implantation (Figure 8a, b, d) as indicated by the GFAP expression level. However, the inflammatory response of both groups becomes weak 4 weeks post-implantation (Figure 8a, b, d). Significantly, the inflammatory response of the Au-BC electrodes (0.31 ± 0.01) is much lower than that of the Au-PI electrodes (1.1 ± 0.03) at 4th week (P

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