Flexible and stretchable opto-electric neural interface for low-noise electrocorticogram recordings and neuromodulation in vivo

Journal Pre-proof Flexible and stretchable opto-electric neural interface for low-noise electrocorticogram recordings and neuromodulation in vivo Bowen Ji, Chaofan Ge, Zhejun Guo, Longchun Wang, Minghao Wang, Zhaoqian Xie, Yeshou Xu, Haibo Li, Bin Yang, Xiaolin Wang, Chengyu Li, Jingquan Liu PII:

S0956-5663(20)30006-3

DOI:

https://doi.org/10.1016/j.bios.2020.112009

Reference:

BIOS 112009

To appear in:

Biosensors and Bioelectronics

Received Date: 28 October 2019 Revised Date:

17 December 2019

Accepted Date: 7 January 2020

Please cite this article as: Ji, B., Ge, C., Guo, Z., Wang, L., Wang, M., Xie, Z., Xu, Y., Li, H., Yang, B., Wang, X., Li, C., Liu, J., Flexible and stretchable opto-electric neural interface for low-noise electrocorticogram recordings and neuromodulation in vivo, Biosensors and Bioelectronics (2020), doi: https://doi.org/10.1016/j.bios.2020.112009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

Credit Author Statement Bowen Ji: Conceptualization, Methodology, Validation, Writing - original draft Chaofan Ge: Methodology, Validation Zhejun Guo: Methodology, Validation Longchun Wang: Validation Minghao Wang: Validation, Writing - review & editing Zhaoqian Xie: Methodology, Validation Yeshou Xu: Methodology, Validation Haibo Li: Validation Bin Yang: Writing - review & editing Xiaolin Wang: Writing - review & editing Chengyu Li: Supervision, Resources Jingquan Liu: Funding acquisition, Writing - review & editing, Project administration

Flexible and stretchable opto-electric neural interface for low-noise electrocorticogram recordings and neuromodulation in vivo Bowen Ji1, Chaofan Ge2,3, Zhejun Guo1, Longchun Wang1, Minghao Wang4, Zhaoqian Xie5, Yeshou Xu6, Haibo Li7,8, Bin Yang1, Xiaolin Wang1, Chengyu Li2,3,*, Jingquan Liu1,*

1. National Key Laboratory of Science and Technology on Micro/Nano Fabrication, Department of Micro/Nano Electronics, Shanghai Jiao Tong University, Shanghai, 200240, China. 2. Institute of Neuroscience and Key Laboratory of Primate Neurobiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China. 3. University of Chinese Academy of Sciences, Beijing, 100080, China. 4. College of Electronics and Information, Hangzhou Dianzi University, Hangzhou 310018, China. 5. Department of Engineering Mechanics, Dalian University of Technology, Dalian, 116024, China. 6. Key Laboratory of C&PC Structures of the Ministry of Education, Southeast University, Nanjing 210096, China. 7. AML, Department of Engineering Mechanics, Tsinghua University, Beijing, 100084, China. 8. Center for Flexible Electronics Technology, Tsinghua University, Beijing 100084, China. *Corresponding authors: Prof. Jingquan Liu, Prof. Chengyu Li Email: [email protected] (J. Liu), [email protected] (C. Li) Tel.: +86 21-34207209; Fax: +86 21-34207209. Address: Institute of Micro/Nano Science and Technology, 800 Dong Chuan Road, Shanghai 200240, P.R. China.

1

ABSTRACT Optogenetic-based neuromodulation tools is evolving for the basic neuroscience research in animals combining optical manipulation and electrophysiological recordings. However, current opto-electric integrated devices attaching on cerebral cortex for electrocorticogram (ECoG) still exist potential damage risks for both brain tissue and electrode, due to the mechanical mismatch and brain deformation. Here, we propose a stretchable opto-electric integrated neural interface by integrating serpentine-shaped electrodes and multisite microLEDs onto a hyperelastic substrate, as well as a serpentine-shaped metal shielding embedded in recording electrode for low-noise signal acquisition. The delicate structure design, ultrasoft encapsulation and independent fabrication followed by assembly are beneficial to the conformality, reliability and yield. In vitro accelerated deterioration and reciprocating tensile have demonstrated good performance and high stability. In vivo optogenetic activation of focal cortical areas of awaked mouse expressing Channelrhodopsin-2 is realized with simultaneous high-quality recording. We highlight the potential use of this multifunctional neural interface for neural applications.

Keywords:

stretchability;

opto-electric

neural

interface;

micro-LEDs;

low-noise;

electrocorticogram recordings; optogenetics

1. Introduction The tremendous advance in neural interfaces facilitate unprecedented understanding of sophisticated brain networks in recent decades, thereby contributing toward the neuroscience research and neurological diseases, such as Parkinson’s disease, Alzheimer’s disease, epilepsy and depression (Buzsáki, 2004; Uhlhaas et al., 2006; Thomas et al., 2015). In electrophysiology monitoring techniques, softer and thinner electrodes are highly desirable for neural surface interfacing (Liu et al., 2015; Guan et al., 2019; Zhang et al.; 2019). As an 2

important class of neural interface for acquiring electrocorticography (ECoG) signals, highly flexible subdural electrode has unique advantages to maintain signal quality over extended periods of time with minimized potential injury to brain tissue (Yeager et al., 2008; Chao et al., 2010). It is also beneficial to realize high-resolution recording over the large-scale cortical surface with abundant information for applications, such as BCI control (Mehring et al., 2003; Viventi et al., 2011). The neural modulation is another vitally important demand for closeloop control of neural activities with simultaneous monitoring. Optogenetics technology is a superior candidate to manipulate specific circuits by optical excitation or inhibition of certain neuron type for functional studies in targeted neural systems (Boyden et al., 2005; Adamantidis et al., 2007). Accordingly, the demand has emerged in the integration of flexible electrode and light source with high resolution. However, the mechanical mismatch between electrodes and neural tissue and the swelling of the brain will not only affect the long-term stability of neural interfaces, but also the safety of ultra-soft brain tissue (Tybrandt et al., 2018; Du et al., 2018). Therefore, traditional flexible electrodes based on polyimide or Parylene-C for ECoG recording are not the optimal choices for long-term implantation in view of the deformation of brain tissue, external pressure from covering and potential damage for the implant or tissue (Kim et al., 2010; Viventi et al., 2011; Khodagholy et al., 2015, 2017; Qiang et al., 2018; Ji et al., 2019). In recent years, further steps have been taken by the development of stretchable electrodes with elastomer substrate (Minev et al., 2015; Yan et al., 2017; Tybrandt et al., 2018; Zhang et al., 2018;) or kirigami structure (Morikawa et al., 2018). Admittedly, the silicone rubber has outstanding stretchability as substrate, but it is difficult to be compatible with micromachining processes like photolithography with high precision. As for the polyimide-based electrode with kirigami structure, problems still exist in the long-term implantation and out-of-plane deformation when stretched. However, as the reliable passivation material to reinforce the metal layer, polyimide is still a good choice with excellent mechanical property and 3

thermostability, as well as micromachining compatibility (Xiang et al., 2016). Consequently, related researchers have proposed the transfer printing method to laminate the polyimide electrode with serpentine layout to the elastomer substrate (Xu et al., 2015; Zhang et al., 2016; Yan et al., 2017; Zhang et al., 2019; Tian et al., 2019). In the way, the flexible electrode is stretchable and durable when implanted on the target tissue. The transfer printing method provides a new path for multilayer stacking, especially meaningful to realize the assembly of flexible electronic devices with stretchability. However, the existing literatures haven’t reported this method with integration of both microelectrodes and micro-LEDs (Kwon et al., 2013; Ji et al., 2018), which are not stretchable with large stiffness. By transfer printing, micro-LEDs can be connected on the polyimide-based electrode and encapsulated by elastomer (Park et al., 2016; Noh et al., 2018). Besides, the micro-LEDs provide unique advantages, including low power consumption, illumination stability, fast light-switching and achievable integration with wireless telemetries (Fan et al., 2015). Here, we provide a solution to the challenge of optoelectronic integration and demonstrate a miniature stretchable opto-electric integrated neural interface (SOENI) with the transfer printing method mentioned above. By means of graphical conductive silver paste and transfer technology by PDMS stamp, the integration of micro-LED chips on polyimide electrode is realized. The polyimide-based photostimulation electrode and recording electrode are separately fabricated with high yield and reliably bonded onto the elastic substrate surface by the condensation reaction between the silicone rubber (Young’s modulus of 166 kPa) and silicon dioxide on the backside of both electrodes. The mechanical, optical, electrical, thermal and electrochemical properties are deeply investigated to illustrate the feasibility and reliability of the device for potential application in long-term implantation. A novel design of serpentine metal shielding layer is added into the recording electrode with no influence on stretchability. It can reduce the electromagnetic interference (EMI) from the micro-LEDs and ambient environment which affects the neural signal quality. The in vitro and in vivo 4

measurements have demonstrated the effect of the metal shielding. In vivo ECoG recording on mouse expressing Channelrhodopsin-2 (ChR2) demonstrates the photostimulation and recording abilities of this multifunctional neural interface. 2. Materials and Methods 2.1 Fabrication of the photostimulation electrode and recording electrode The recording electrodes and photostimulation electrodes were individually fabricated on two wafers with similar MEMS processes. The main fabrication processes are described as below: Two 4-inch silicon wafers were rinsed by RCA standard cleaning method. A 300 nm thick aluminum (Al) as sacrificial layer was evaporated on both wafers by physical vapor deposition, which would benefit the release of flexible electrodes from wafer. Spin casting of photosensitive polyimide (Durimide 7505, Fujifilm, Japan) with speeds of 1500 rpm and 3000 rpm was applied on two wafers, respectively. After soft baking, photolithography, development in HTRD2 (Fujifilm, Japan) and rinse in RER600 (Fujifilm, Japan), the patterned 1st PI layer was finally cured in N2 at 300 ˚C for 1 hour to form approximately 5 µm and 2.5 µm in thickness, respectively. The lower curing temperature contributed to leave unterminated bonds and better cohesion with followed polyimide layer. Then, electron beam evaporation formed a thin (30/300 nm) layer of Cr/Au on both wafers followed by spin casting of 3 µm thick positive photoresist (AZ P4330, AZ Electronic Materials, USA) and photolithography. Next, the ion beam etching system was used to realize dry etching of metal film in argon atmosphere. The patterned metal layer stood as the conducting layer for photostimulation electrodes and the electromagnetic shielding layer for recording electrodes. For the stimulation electrodes, another 5 µm thick PI layer was spun, photoetched and cured in N2 at 350 ˚C for 1 hour as the encapsulation layer. For the wafer with recording electrodes, the 2nd PI layer was spun, patterned and cured in N2 at 300 ˚C for 1 hour as the insulation layer with thickness of 2.5 µm. The same metal 5

evaporation, photolithography and etching processes were conducted to obtain the 2nd metal layer comprising Cr/Au (30/300 nm) as the electrode layer. Finally, the 3rd PI layer was spun, patterned and cured in N2 at 350 ˚C for 1 hour as the encapsulation layer with thickness of 2.5 µm. 2.2 Sample releasing from silicon wafer In general, the flexible PI-based devices can be directly immersed in dilute hydrochloric acid solution for releasing. The intact whole piece of PI film may only curl slightly, but the serpentine-shaped PI devices are extremely easy to be self-cured and tangled together as shown in Fig. S2C. Thus, a unique technique of pressure-assistant release was applied to keep the released serpentine device in its original shape. The wafer was firstly covered by 3 layers of air-laid papers and a 4-inch glass wafer, and two clips were used to clamp the stacked wafers on edges. Immerse them in 20% HCl solution for 24 hours to finish the slow process of metal sacrifice (Al), followed by rinsing in deionized water and fully drying in 100 ˚C oven for 2 hours in the clamping state. After releasing the clips, water-soluble adhesive tape (polyvinyl alcohol, PVA, Aquasol, North Tonawanda, NY) easily retrieved PI electrodes from the silicon wafer as shown in Fig. S2D. The magnetron sputtering systems deposited 5 nm of Ti and 50 nm of SiO2 onto the backside of electrodes for adhesion to silicone substrates in the subsequent assembly steps. 2.3 Assembly of the SOENI device The main assembly processes included spinning two layers of near-transparent silicone rubber, transfer printing of the photostimulation electrode and recording electrode on PVA tape in sequence, transfer printing of the micro-LED array to the photostimulation electrode on the customized platform, brushing patterned conductive silver paste for cable connection, interface sealing, laser cutting and electrochemical modification. The detailed assembly flows were described in the Supporting Information (Note S1). 2.4 Tensile testing 6

The tensile test bench consists of four independent stepping motors with ball screws and 3D-printed clamps in biaxial directions, a programmable controller, driver circles, a customized baseplate and a stereoscopic microscope, as shown in Fig. S6A. Due to the relatively small size of the device, it was adhered on a Dragonskin carrier (90×50×3 mm3) for easy clamping on both ends Fig. S6B. The adhesion was realized by the silicone rubber adhesive (Sil-Poxy, Smooth-on, USA), which has strong adhesion for bonding RTV silicone rubber to silicone rubber, great stretchability and tear resistance with cure time of 12 min at room temperature. The unidirectional reciprocating speed was set as 3 mm/s with high accuracy. 2.5 Mechanical finite-element analysis ABAQUS commercial software (ABAQUS Analysis User’s Manual 2010, V6.10) was used to study the mechanics properties of the assembled SOENI device under two deformations (10% stretching and bending to R=4 mm). The Mooney-Rivlin model was used to represent the Dragonskin substrate with temperature-dependent material parameters: C10=E/5(1+ν), C01= E/20(1+ν) and D1=6 (1-2ν)/E. Linear elasticity was used to define the PI film and the elastic-plastic model was used to model the Au and SiO2 layers. The detailed Young’s moduli and Poisson's ratios used for FEA are summarized in Table S1. The Dragonskin

substrate

was

modeled

by the

hexahedron

element

(C3D8R).

The

photostimulation electrode embedded in the Dragonskin substrate was set as the skin. The recording electrode, which consisted of a Ti/SiO2 adhesion layer, three PI layers and two Au layers, was modeled by four-node composite shell elements (S4R). The meshes used in the numerical calculation were refined to ensure accuracy. The elastic-plastic transition is set when the maximum strain of half the width of one section is beyond the yield strain of 0.3%. 2.6 Optical characterization Due to the relatively weak illumination by micro-LED chip on the device, its radiant flux was measured by a specialized integrating sphere (FOIS-1, Ocean Optics, USA), a 7

spectrometer (Flame, Ocean Optics, USA) and an optical fiber for connection, as shown in Fig. S8B. In order to test the light intensity more accurately, the integrating sphere was placed upside down to cover the sample. Before the optical testing, the optical compensation file should be loaded into the software (Ocean Optics SpectraSuite) with integral time of 3 s, average times of 50 and smoothness of 5. Meanwhile, the dark noise removal and stray light correction options should be ticked. Four samples were tested before and after soaking and repeated stretching to verify the optical characterization. 2.7 Thermal characterization by thermal imager The temperature rise at the top surface of Dragonskin above the micro-LED chip was measured by a thermal imager (Fotric 228s, FOTRIC, USA) with thermal sensitivity (NETD) of 0.03 ˚C. The macro lens (M20-228s, FOTRIC, USA) was used for high-resolution measurement of area down to several hundreds of microns. Fixed on the universal test bench, the macro lens faces down to the sample, which is attached on the agar gel to imitate the brain tissue. Three close measurement points were picked right above the area of one micro-LED for comparison to choose the highest temperature output value. The multi-channel power supply (SEMIPIN uPEony-12, SEMIPIN PTE. Ltd., Singapore) was used to drive the microLED with different parameters of voltages, duty cycles and frequencies in the study of temperature rise. 2.8 Thermal finite-element analysis Finite-element modeling was also implemented with the commercial software ABAQUS to study the changes of temperature in brain tissue that is contacted with the device. To reduce computing scale, the device model was simplified as partial photostimulation electrode with micro-LEDs embedded two Dragonskin layers, not including the relatively thin recording electrode on top surface. The brain tissue and device were modelled by hexahedral element (DC3D8), with proper minimal mesh size to ensure the convergence of simulation. The

8

detailed thermal conductivity, heat capacity and mass density used for FEA are summarized in Table S2. 2.9 Electrochemical characterization The electrochemical experiments of the electrodes were conducted on a PGSTAT12 Autolab workstation (EcoChemie, Utrecht, Netherlands) with a conventional three-electrode setup, with the SOENI as the working electrode, the Pt sheet as the counter electrode and the saturated calomel electrode as the reference electrode in phosphate-buffered saline (pH 7.2 to 7.4) at room temperature. CV was scanned over the potential range of -0.6 V and 0.8 V at the scanning rate of 100 mV/s. EIS experiments were performed at the frequency range of 0.1 Hz to 100 kHz with AC excitation voltage amplitude of 10 mV at open circuit potential (OCP). Four samples were tested before and after soaking and repeated stretching to illustrate the stability and uniformity of Pt-black modified electrodes corresponding to Fig. 4G. The charge storage capacity (CSC) was calculated from the area obtained by integrating the current with E respect to voltage scan rate as CSC = 1 ∫ i dE , where E is the electrode potential, i is the E a

v

c

S

measured current, S is the geometric area of the microelectrode site, v is the scan rate (V/s), and Ea and Ec are the anodic and cathodic potential limits, respectively. 2.10 In vivo animal experiments All experiments are approved by the Animal Care and Us Committee of the Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China. VGAT-Cre transgenic mouse (12 weeks, weighted 25 g) expressing AAV-DIO-ChR2 virus was used for the acute experiment. An awake mouse was head-fixed by a plate to the fixing bar. A rectangular craniotomy was made with a surgical drill and the bone flap was separated with a microdissector, leaving the cerebral dura mater exposed. Approximately 4×3 mm2 cranial window was made exposing the area across the somatomotor cortex and somatosensory cortex. The SOENI was placed on the epidural cortical surface and kept in good state with a 9

slurry of gelfoam and artificial cerebrospinal fluid (ACSF). A stainless-steel screw electrode was implanted in the drill holes, with the reference position at cerebellum (Cb), where exhibits lower activity compared to other brain sites. The 9-electrode location is shown in Fig. 6B. Experiments were conducted in a well-lit and shielded room. Once experimentation was completed, the mouse was killed immediately with an intraperitoneal injection of pentobarbital solution. ECoG signals from nine channels were amplified and digitized at a sampling rate of 1 kHz with the Multichannel Acquisition Processor (Plexon Inc, USA) along with optical stimulation by integrated micro-LEDs. The data were processed in EEGLAB package with frequency pass band of 0.5~200 Hz. 3. Results and Discussion 3.1. Design concept and device structure The stretchable opto-electric integrated neural interface (SOENI) is made to match the area of somatomotor cortex and somatosensory cortex of mouse brain. Considering the relatively compact design space for electrode distribution, 3×3 microelectrode sites (100 µm in diameter), 2×2 micro-LEDs (180×230×50 µm3) and one large reference electrode (250 µm in diameter) are arranged as shown in Fig. 1A and its enlarged view in Fig. 1B. The SOENI consists of the photostimulation electrode embedded in two silicone elastomer layers and the recording electrode attached on the surface of top elastomer layer, in which micro-LEDs are connected by flip-on-chip method with their illuminated surfaces on the same side with microelectrode sites. The whole device size including front-end microelectrodes and connection leads is 12.7×3.6 mm2 as illustrated in the Supporting Information (Fig. S1). All interconnecting wires for both photostimulation and recording electrodes are designed as serpentine shape to improve the stretchability with internal angle of 225˚, line width of 50 µm and radius of 100 µm. Four micro-LEDs are surrounded by the nine recording microelectrode sites, with four microelectrode sites distributed uniformly on the four corners of every microLED. The pitch between microelectrode sites is 700 µm, and same for the micro-LEDs. Thus, 10

the relative distance between any microelectrode site and adjacent micro-LED is 350 µm in both x and y directions. This structure design gives consideration to the lithography precision of mask, the micromachining cost, the limit of device size and the stretchability as large as possible. The whole device is composed of 13 layers as pictured in Fig. 1C, mainly consisting of two elastomer layers made of Dragonskin (10 Slow, components A:B=1:1, Smooth-on, USA) as the substrate (bottom) and encapsulation (top) layers, photostimulation electrode and recording electrode with Ti/SiO2 deposited on both backsides and four micro-LEDs. The photostimulation electrode includes two polyimide (PI) layers, Cr/Au metal layer and the Ti/SiO2 adhesion layer. The recording electrode comprises three PI layers, two Cr/Au metal layers and Ti/SiO2 adhesion layer. As a high-quality platinum catalyzed silicone rubber, Dragonskin has much lower Young’s modulus (166 kPa) than polydimethylsiloxane (PDMS, 10:1, 1 MPa) which is more suitable for the flexible and elastic neural interface. The assembled SOENI is pictured in Fig. 1D and its enlarged view in Fig. 1E, with satisfied assembly result as we expected. The disconnected device was attached conformally on a wide oval yellow Kerria flower petal as shown in Fig. 1F illustrating its flexibility. 3.2. Fabrication processes The photostimulation electrodes and recording electrodes are separately fabricated on silicon wafers (Fig. S2A and S2B). To avoid tangles during device releasing (Fig. S2C), a unique technique of pressure-assistant release and retrieve by polyvinyl acetate (PVA) tape was applied to keep the released serpentine device in its original shape (Fig. S2D). The detailed fabrication processes are described in Materials and Methods. In the following assembly processes (Fig. S3), a key step is transfer printing multiple micro-LEDs by the PDMS stamp. Reversed from convex SU-8 mold (Fig. S4A), the concave PDMS stamp has a depth of 25 µm to position micro-LEDs with height of 50 µm (Fig. S4B). With the aid of customized three-axis moving platform, the micro-LEDs were applied to the receiving substrate with patterned paste (Fig. S4C). After the heating module worked on wafer at 80 ˚C 11

for 10 min, the PDMS stamp was slowly peeled back, leaving all micro-LEDs printed onto the receiver successfully, which can be activated sequentially (Fig. S5C). From the fabrication results in the Supporting Information (Fig. S5A and B), the serpentine-shaped photostimulation electrode is in good condition with independent anodes and common cathode for micro-LEDs. The transferred micro-LED is located at the area of rectangle PI substrate, which is applied as an island for stable connection. As for the recording electrode, the two metal layers are vertically well-aligned on microelectrode sites and serpentine leads, and the EMI pad is kept to the side with ten electrode pads arranged with pitch of 0.5 mm (Fig. S5D to G). The complete assembly processes are described in the Supporting Information (Note S1). Single micro-LED can be driven with different supply voltages and frequencies on the final assembled device (Movie S1). Besides, the cycle operation of micro-LED with various modes and multiple ones operating at the same time can also be independently controlled (Movie S2 and Movie S3). One more severe problem is the adhesion strength of the two-layer Dragonskin films. When releasing the SOENI device by a tweezer after laser cutting (Fig. S6A), delamination was easily occurred between the top and bottom Dragonskin layers without any pre-treatment on the interface (Fig. S6B). To avoid permeation of liquid and ensure reliable encapsulation for micro-LEDs, the bottom Dragonskin was pretreated with O2 plasma for 1 min due to the activation of silicone rubber surface with strong Si-O-Si chemical bond at the interface. No delamination was observed at two Dragonskin layers which could withstand severe stretching deformation (Fig. S6C). To further validate the adhesion strength, two plasma-treated samples were immerged in the thermostatic 75 ˚C water bath for 3 days and 7 days (Fig. S6D). Satisfactorily, no delamination was observed in the SEM for both samples compared to the one without O2 plasma treatment after severe stretching (Fig. S6E to G). 3.3. Mechanical properties of stretching and bending

12

The SOENI device will inevitably suffer from deformations including pressing, stretching and bending during implantation on the cortical surface caused by manual operation or coverings like dental cement. Besides, the swelling of brain tissue will also affect the device after implantation. Thus, the mechanical properties of device need to be evaluated with different deformations. Although Ti/SiO2 stands as the adhesion layer for the PI electrode and silicone rubber Dragonskin, the debonding risk still exists under deformation to some extent, especially for the recording electrode which is attached on the top Dragonskin surface. Stretching of the SOENI was realized with the help of a large Dragonskin carrier on the tensile test bench as described in Materials and Methods. The initial length of the carrier is 75 mm. When the carrier was stretched to 82.5 mm (10% strain), all serpentine wires deformed with the substrate and kept in bonding state. However, as the length increased to 90 mm (20% strain), about 1/3 wires are partially debonded from the substrate and regarded as failure for the device as shown in Fig. 2A. About 2/3 unrecoverable serpentine wires were detached from the surface after relaxing the carrier from 30% stretching deformation to original length as shown in Fig. S7D. No such high deformation is needed for practical applications. Thus, deformation of 10% can meet the requirement on most occasions.[20] Cyclic stretching from the initial state to 10% deformation for hundreds of times indicates that no debonding has been observed from the substrate. The stretching performance of SOENI was further analyzed by finite element analysis (FEA) in ABAQUS. The displacement deformation of SOENI device is 1.18 mm in y-axis direction with initial length of 19.5 mm when the carrier is stretched to 10% (Fig. S7C). As shown in Fig. 2B, according to the strain distribution in the Au layers derived from FEA results, no elastic-plastic transition occurs in photostimulation electrode (left) or recording electrode (right) with 10% deformation. Besides, the Au layer in recording electrode suffers from larger stress than the one in photostimulation electrode. FEA results (Fig. 2D) corresponding to experiment (Fig. 2C) demonstrate that the maximum principal strain in the Au layer does not reach the yield strain value (0.3%) upon 13

bending on a transparent tube with the radius of 4 mm. These experimental and FEA results indicate that the mechanical properties of as-fabricated device facilitate the long-term implantation on the brain tissue. 3.4. Optical, electrical and thermal properties Considering the lower transparency of Dragonskin than PDMS, the transmittance of micro-LED through Dragonskin film needs to be measured. Four film samples are compared with thickness of 50 µm, 100 µm, 200 µm and 400 µm as shown in the Supporting Information (Fig. S8A). The film of 50 µm is too soft to be spread out in flat state, which is not suitable as the substrate of the device. In addition, it is too thin as encapsulation for microLED chips with the height of 50 µm as well. As the thickness increases, the light transmittance gradually attenuates. To measure the radiance of micro-LED accurately, a specialized integrating sphere connected to a spectrometer by optical fiber is used (Fig. S8B). The transferred micro-LED chips on the sample is naked without following encapsulation process for the optical measurement. By covering the naked micro-LEDs with the Dragonskin films of varied thicknesses (50-400 µm) (Fig. S8C), the peak value of radiance at wavelength of 468.8 nm is gradually reduced with the increase of film thickness in Fig. 3A. The percent of light transmitted through Dragonskin films of different thicknesses was assessed by measuring the power of blue light from micro-LED at wavelengths of 440 nm, 460 nm, 468.8 nm (peak point), 480 nm and 500 nm (Griggs et al., 2019). Taken the uncovered micro-LED as 100%, the light transmittance values at peak wavelength are 98.8%, 97.1%, 94.5% and 90.4% for the films with the thickness of 50 µm to 400 µm, respectively (Fig. 3B). The thickness of the top Dragonskin encapsulation layer is only 100 µm, therefore, it is about 50 µm above the micro-LED in view of self-leveling of Dragonskin before curing. It is believed that a thin Dragonskin film of 100 µm has little influence on the light transmittance. The stability of output optical power of encapsulated micro-LEDs is another key question for the SOENI. 14

The light power of single micro-LED on the device was also measured by the integrating sphere with constant voltages changed from 2.6 V to 3.4 V. The output light power is varied from 0.10 mW to 1.95 mW with the corresponding power intensity of 3.1 mW/mm2 to 60.0 mW/mm2, which meets the requirement of minimum power intensity of 1 mW/mm2 to induce neuronal action potentials (Boyden et al., 2005). To further study the stability of optical performance, four samples with 16 micro-LEDs were used for testing, in which two of them are soaking in 75 ˚C PBS for 7 days and the other two are under 10% stretching for 500 cycles. The averaged output light power with standard deviation are compared before and after soaking and stretching when the voltage changes from 2.6 V to 3.4 V as shown in Fig. 3C. The slight optical power attenuation after soaking or stretching proves that the as-fabricated SOENI is suitable for long-term implantation even undergoing liquid immersion and deformation. The same four samples were also tested in electrical properties before and after soaking and stretching with a semiconductor parameter analyzer (BA1500, Agilent Technologies, USA). The nonlinear current-voltage (I-V) curves are pictured in Fig. 3D with standard deviation at the voltage of 2.6 V, 3.0 V and 3.4 V. Taken voltage 3.4 V as an example, the standard deviations of current are 0.95 mA, 1.13 mA and 1.30 mA at initial state, after soaking and stretching, respectively. The cyclic stretching has greater influence on the consistency of electrical performance than accelerated aging test by soaking for the assembled micro-LEDs in device. Besides, the unobvious current attenuation from I-V curves proves the stability of electrical properties. The insets of Fig. 3D illustrate the uniform luminance of four micro-LEDs under the voltage of 2.6 V, 3.0 V and 3.4 V. In practical optogenetics application, the brain tissue damage caused by the heat production of micro-LED is a major concern. The heat generated in the micro-LED and emitted light are the main sources and the heat transmission from the micro-LED package to brain tissue occurs through bioheat transfer (Henry et al., 2018). Accordingly, the temperature 15

variation of single micro-LED in the device was investigated at varied duty cycles (0-25%), voltages (2.6-3.4 V) and frequencies (5-40 Hz) with a thermal imager and macro IR lens. The temperature goes up and stabilizes for operation with the micro-LED on and quickly drops back to the baseline when it is off with duty cycle of 25% and frequency of 5 Hz (Fig. 3E). Obvious difference can be found for the sample under different voltages. The averaged maximum rise in temperature changes with duty cycle is illustrated in Fig. 3F as functions of voltage and frequency. The temperature rise grows with the increase of both duty cycle and voltage distinctly and drops with the frequency increasing. With highest duty cycle of 25% and voltage of 3.4 V and lowest frequency of 5 Hz, the temperature rise of 1.03 ºC is largest for all testing parameters, which satisfies the maximal acceptable temperature rise 1 ºC for brain tissue (Childs, 2008; Gutruf et al., 2018). It needs to be emphasized that the practical duty cycle is usually below 5% with enough response time for neurons. In addition to the thermal conduction through the metal and paste below the micro-LED, the brain tissue also helps to dissipate heat via conduction and blood perfusion (Wu et al., 2015). Also, the temperature rise of the brain is expected to be slower than the micro-LED surface due to the higher thermal conductivity of brain tissue than the air with an order of magnitude. The finite element thermal analysis provides further insights into the temperature rise and distribution in the brain. The cross-sectional and vertical schematic illustration of the simplified model exhibits the dimensions and the spatial location of the output temperature spot contacting with the brain tissue as shown in Fig. 3G. Act as independent heater, every micro-LED is input with thermal power and time function to realize interlaced, pulsed mode operation with frequency of 20 Hz, voltage of 3.0 V and duty cycles of 5% and 25% for comparison (Fig. 3H). The corresponding calculated peak value of input thermal power is 7.7 mW with 2000 data/sec for all output nodes in the modelling to study the temperature rise with time. To avoid oversize output files, the temperature output area is picked as a small area at Dragonskin top surface over the corresponding micro-LED as shown in Fig. S9A. The 16

temperature change at four output temperature spots adjacent to brain tissue is plotted in Fig. 3I, with stable maximum temperature rise of 0.12 ºC and 0.38 ºC for respective duty cycle of 5% and 25% at four seconds. The temperature distribution of four output areas is also illustrated in Fig. S9B with micro-LEDs at stable maximum temperature in order. The temperature rise distributes inconsistently from the bottom to the top of rectangle output area, with the largest temperature rise of 0.6 ºC close to the working micro-LED and 0.38 ºC adjacent to brain tissue. Replace the temperature output area to the brain model, it is clear to find out the temperature rise and distribution in the brain tissue from Fig. 3J with duty cycle of 25%. 3.5. Electrochemical properties To enhance signal-to-noise ratio (SNR) and reduce resistance, the microelectrodes are electrochemically modified by ultrasound-assisted deposition of platinum black (Pt-black). All microelectrode sites were cleaned by ultrasonic cleaning in DI water, O2 plasma treatment with nanoscale rough Au surface and CV scanning from -1.0 to 1.0 V beyond the water window before electroplating Pt-black. A repetitive current pulse train (5:500 ms duty ratio, 4.5 A/cm2 peak current density, 300 cycles) was applied in chloroplatinic acid solution (H2PtCl6) with a gold rod as both counter and reference electrodes in ultrasonic bath (50 W, 40 kHz) by electrochemical workstation (CHI660c, CH Instrument, China). From the full view of modified microelectrodes in Fig. 4A, all sites are uniformly electroplated without delamination or cracks. Furthermore, comparison is made for the same microelectrode site before and after deposition of Pt-black (Fig. 4B). The SEM pictures show clear uniformly distributed cauliflower-like 3D microstructure of Pt-black coating with high effective surface area (Fig. 4C), which facilitates the long-term neural signal recording with excellent performance. The plots in Fig. 4D, E and F illustrate the cyclic voltammetry curves, electrochemical impedance and phase in all nine channels of a sample. CV, impedance and phase for each channel are close to each other with good consistency, indicating the ability for 17

multichannel electrical activity recording from the SOENI. To make better validation of the stability of Pt-black coating, four samples were prepared with the same electroplating parameters. Two samples were soaked in 75 ˚C PBS for 7 days, and the other two were repeatedly stretched with 10% deformation for 500 cycles. All microelectrode sites were measured and calculated with average and standard deviation for the initial, soaking and stretching states (Fig. 4G). The charge storage capacity (CSC) are 114.9±17.7 mC/cm2, 109.0±20.3 mC/cm2 and 103.9±25.8 mC/cm2 for these three states, respectively, with little attenuation after soaking or stretching. The impedances at 1 kHz are 6.72±0.96 kΩ, 6.78±1.02 kΩ and 6.99±1.25 kΩ and the phases at 1 kHz are -3.17±0.80˚, -3.42±0.85˚ and -3.88±0.99˚ for the three states. As can be seen, the impedance only increases by 0.9% and 4.0% after soaking and stretching, respectively, and the phase changes little as well. 3.6. Serpentine metal shielding for EMI Active micro-LEDs and their interconnects will introduce electromagnetic interference (EMI) to the recorded signals with artifacts which may obscure or distort neural activities (Kampasi et al., 2018; Guo et al., 2019). In order to improve the quality of signals, a layer of metal shield is added into the recording electrode to weaken the effect of EMI. We have compared four cases with different metal shield structures for electrostatic field simulation, including the one without shield, the one with serpentine shield, the one with sheet shield and the one with farther serpentine shield as illustrated in Fig. 5A. In the simulation, the potential at metal shield layer is set as zero which is regarded as grounding. A section of serpentine wire is used as the model for simulation in COMSOL software (Fig. 5B). The whole wire is composed of three layers of PI and two layers of metal Au with the top as the recording metal layer and the bottom as the shielding metal layer. The electrostatic field with any voltage is added above the wire and the potential distribution of the wire is normalized for the comparison of four cases as shown in Fig. 5C. Four cases with the normalized potential distribution in the cross section of serpentine wire are compared in Fig. 5D. The potential at 18

the recording metal layer drops off dramatically compared with the one without any shield. The sheet shield has the best shielding effect, but it cannot satisfy the demand of stretchability with the whole device. The farther serpentine shield, with distance of 5 µm to the recording metal layer, is not as effective as the serpentine shield with distance of 2.5 µm. To illuminate quantitatively the EMI effect, the normalized potentials at recording metal layer are plotted along the width direction for all cases as plotted in Fig. 5E. Specifically, the normalized potentials are 0.493 for the one without shield, 0.103 for the one with serpentine shield, 0.002 for the sheet shield and 0.252 for the farther serpentine shield, respectively. Accordingly, the serpentine metal shield layer with the distance of 2.5 µm to the recording metal layer has good shielding effect for almost 90% EMI. To valid the metal shielding effect in the SOENI, the in vitro tests are firstly conducted in PBS (0.1 M). The SOENI is immersed in the solution with its long PI cable inserted into the FPC connector on PCB with the Omnetics connector on the other end, to an RHD2164 amplifier board connected to an RHD2000 Evaluation System (Intan technologies, Los Angeles, CA, USA) as shown in Fig. 5F. The ground wire of amplifier is also immersed in the solution. The channel for shielding is separated from other channels on the PCB for the convenience of on-off switching to the ground. One of the micro-LEDs is flashing with the voltage of 3.0 V, frequencies of 1 Hz, 5 Hz and 20 Hz, and duty cycles of 5% and 25%. The evoked artifacts from the recording microelectrode site close to the working micro-LED are compared with and without metal shielding grounded as shown in Fig. S10. The amplitude of evoked transient artifacts reduces with the increase of frequency. Meanwhile, the SNR of the device with metal shielding grounded is higher than the one ungrounded, and the amplitude decreases exceeding 50% compared with the ungrounded one. When the duty cycle changes from 5% to 25%, the artifact amplitude increases as well, illustrating the influence of frequency and duty cycle on the artifact amplitude besides the supply voltage. To further figure out the artifact with one light pulse, comparison is made between the one with and 19

without grounding and duty cycle of 5% and 25% (Fig. 5G). For the one with duty cycle of 5%, a small forward peak pulse appears at the start moment of power supply, and then the waveform drops dramatically with a negative spike followed by returning to the baseline slowly. With the metal shielding grounded, the amplitude of baseline noise decreases from 400 µV to 175 µV, and the waveform has smaller peak-to-peak value and recovers faster than the ungrounded one after the pulse. When the duty cycle rises to 25%, the waveform dropped with the light duration of 250 ms and the recovery time is longer than the one with duty cycle of 5%. The peak-to-peak voltage decreases after the metal shielding grounded apparently and recovers faster to the baseline level. The averaged peak-to-peak voltages of evoked artifacts from Fig. S10 are compared at different photostimulation frequencies and duty cycles with and without shielding metal layer grounded (Fig. 5H). The peak-to-peak value for duty cycle of 5% and 25% deceases by 63.8% and 57.7% at 1 Hz, 54.6% and 63.6% at 5 Hz, 53.9% and 55.5% at 20 Hz after grounding. Thus, the introduce of shielding metal layer can reduce the impact of EMI from the micro-LED especially when grounded. 3.7. In vivo ECoG recording and optical stimulation The acute animal experiment is conducted on Thy1-ChR2-YFP mouse to valid the photostimulation and recording abilities of SOENI. The surgically related details can be referred from in Materials and Methods. Thank to the low modulus of the device, no damage to the brain tissue was occurred with repetitive adjustment of its position and attachment for many times during the experiment. Fig. 6A and B show the illustration and photo of the SOENI attached on the cortical surface with optical stimulation from embedded micro-LED, respectively. In the animal experiment, a set of stimulating parameters are used for single micro-LED as follows: voltage of 3.0 V (corresponding irradiance of 26.7 mW/mm2), frequency of 5 Hz, duration of 5 ms and 10 pulse cycles (Movie S4). The ECoG signals recorded from all channels are plotted without (shown above) and with (shown below) metal shielding grounded in Fig. 6C. It shows neural response with negative potential to the optical 20

stimulation in each channel with diverse amplitudes due to the distance difference of microelectrode sites to the working micro-LED. The SNR of ECoG signals is significantly improved after grounding. Therefore, the shielding metal layer can inhibit the EMI form the surrounding environment, and the switching of micro-LED does not cause significant EMI change to the neural signal. The introduction of metal shielding is quite necessary. It can play a role in inhibiting the EMI and improving the SNR, which is conducive to distinguishing the light-induced neural signal response from the baseline. The signals from channel 1 are enlarged and compared in Fig. 6D. It is clear to find out the low-noise ECoG signal baseline after grounding with neural response to light pulses. The most important function of the SOENI is the simultaneous photostimulation and recording with high spatial resolution. Here, two micro-LEDs at diagonal positions are selected to work separately with the same stimulating parameters. The negative potential response induced by a single pulse (duration of 5 ms) is analyzed at the same time. As shown in Fig. 6E, LED1 is in the center of ch1, ch2, ch3 and ch6, which induces higher negative potential amplitude of more than 500 µV. As comparison, the relatively further microelectrode site labelled ch7 owns the potential amplitude of less than 200 µV. When the working micro-LED changes to the position of LED3, the magnitudes of evoked negative potentials in all channels are different from the ones with LED1 on (Fig. 6F). The similar distribution regulation of the amplitudes in all channels demonstrates the good spatial resolution of the SOENI. In the future work, long-term implantation will be conducted to study the influence of optogenetics to the animal behaviors and neural activities, considering the issues of potential infection, subsequent inflammation and biocompatibility. 4. Conclusion In summary, we have introduced a flexible and stretchable opto-electric integrated neural interface in this work to realize simultaneous photostimulation and recording on the cortical surface of mouse with high resolution. Starting from the thorny problem of mechanical 21

mismatch between electrode and neural tissue and the swelling of the brain, the proposed device maintains good performance after cyclic stretching, which is vitally important to the potential application in the long-term implantation for clinical practice and neuroscience research. Furthermore, low-noise recording can be realized by the introduce of serpentineshaped shielding metal layer, which can reduce the impact of EMI from the micro-LED and ambient environment and has negligible influence on the stretchability. However, the adhesion strength between polyimide-based electrodes and silicone substrate needs further improvement to prevent debonding with more severe deformation. In the future, the SOENI will benefit from the introduction of thinner, smaller and more flexible micro-LEDs and more compact microelectrode sites for high-resolution neural modulation. Besides, this neural interface is expected to be useful in the optogenetics application on other organs or tissues, such as heart, cochlea, spinal cord and peripheral nerves from mice to non-human primates. Acknowledgements The authors gratefully acknowledge the support of the National Key R&D Program of China under grant 2017YFB1002501, the National Natural Science Foundation of China (No. 61728402), Research Program of Shanghai Science and Technology Committee (17JC1402800),

Program

of

Shanghai

Academic/Technology

Research

Leader

(18XD1401900), National Postdoctoral Program for Innovative Talents (BX20190174). The authors are grateful to the Center for Advanced Electronic Materials and Devices (AEMD) of Shanghai Jiao Tong University, Dr. Xiaowei Gu’s suggestion in animal experiments from RIKEN Brain Science Institute, as well as Prof. Yonggang Huang’s joint supervision when B. J. stayed in Northwestern University.

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FIGURES

Fig. 1. Stretchable opto-electric integrated neural interface (SOENI) for cortical mapping and neuromodulation. (A and B) Schematic diagram of the flexible SOENI integrated with microLEDs and recording microelectrodes. (C) Layout of the proposed SOENI device. (D) Photograph of the SOENI with any single micro-LED on. (E) Photograph of the serpentine structure design corresponding to the diagram in (B). (F) SOENI device attached on the curved surface of Kerria petal.

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Fig. 2. Mechanical characteristics of the device. (A) Photographs of the device glued on the large-area thick Dragonskin substrate from initial state to 20% stretching along one direction. Scale bar:100 µm. (B) The FEA strain distribution of Au metal layer in the photostimulation electrode and recording electrode of the SOENI under 10% uniaxial stretching. (C) Photograph of the device when bending on a tube with radius of 4 mm. (D) The FEA strain distribution of Au metal later in the assembled device corresponding to the photograph in (C).

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Fig. 3. Optical, electrical and thermal properties. (A) Radiance and (B) light transmittance curves of bare micro-LED chip covered by Dragonskin films of different thicknesses. (C) Comparison of the output light power of samples at initial state, soaking in 75 ˚C PBS for 7 days and repeated 10% stretching for 500 cycles at different voltages from 2.6 V to 3.4 V. (D) The I-V curves of samples at initial state, soaking in 75 ˚C PBS for 7 days and repeated 10% stretching for 500 cycles. Insets illustrate the luminance of four micro-LEDs under different voltages. (E) Temperature measurement of single micro-LED operating at 5 Hz and 25% duty cycle under voltages of 2.6 V, 3.0 V and 3.4 V. (F) Measured temperature rise of brain tissue adjacent to the micro-LED with different duty cycles, voltages and frequencies. (G) Crosssectional and vertical schematic illustration of the model used for finite element thermal analysis. (H) Plot of thermal power as a function of time for each of micro-LEDs during interlaced, pulsed mode operation (20 Hz), with 5% and 25% duty cycles, respectively. (I) Plot of the maximum change in temperature of the brain tissue as a function of time for operational conditions shown in frame (H). (J) 3D cutaway and X-Y plane illustration of 3

temperature change distribution in brain tissue, for steady state operation after 4 seconds at 25% duty cycle, 3.0 V and 20 Hz.

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Fig. 4. Electrochemical properties. (A) The full view of microelectrodes with electrochemical deposition of Pt-black. (B) Comparison of single microelectrode site before (the Fig. above) and after (the Fig. below) Pt-black deposition. (C) SEM pictures of single microelectrode site with its partial enlarged view of cauliflower-like Pt-black in red dotted box. (D) cyclic voltammetry curves, (E) electrochemical impedance spectra and (F) phase curves measured at nine microelectrode sites conFig.d for ECoG recording. (G) Comparison of CSC, impedance and phase at 1 kHz for microelectrodes of initial state, soaking in 75 ˚C PBS for 7 days and repeated 10% stretching for 500 cycles.

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Fig. 5. Electrostatic field simulation and in vitro test of stimulation artifacts with serpentine metal shielding. (A) Sectional illustration of recording electrode wire with four different metal shielding structures for simulation. (B) Three-dimensional structure diagram of the serpentine wire. (C) Normalized potential distribution of the serpentine wire in electrostatic field simulation. (D) Comparison of normalized potential distribution in the cross section of recording electrode wire between the four different metal shielding structures. (E) Comparison of normalized potential curves along the width of recording metal layer between the four different metal shielding structures. (F) Measurement setup for in vitro noise characterization with assembled micro-LED flashing in PBS. (G) Comparison of evoked artifacts with and without shielding metal layer grounded, along with single micro-LED working at voltage of 3.0 V, frequency of 1 Hz and duty cycles of 5% and 25%. (H) Comparison of peak-to-peak voltages of evoked artifacts at different frequencies and duty cycles, with and without metal shielding layer grounded.

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Fig. 6. In vivo recording of ECoG signals with optogenetics stimulation. (A) Illustration and (B) photo of the SOENI on the cortical surface of mouse with optical stimulation. (C) Comparison of ECoG signals recorded from all 9 channels with optical stimulation pulses (voltage of 3V, frequency of 5 Hz and duration of 5 ms) without (shown above) and with (shown below) metal shielding grounded. (D) Comparison of ECoG signals of ch1 without (shown above) and with (shown below) metal shielding grounded. (E and F) Evoked negative potentials in all channels by single light pulse (5 ms) at LED1 and LED3.

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Highlights 1. A stretchable opto-electric integrated neural interface was proposed by integrating serpentine-shaped bioelectrodes and multisite micro-LEDs onto hyperelastic substrate with satifying stretchablility. 2. A serpentine-shaped metal shielding layer was embedded in recording electrode with negligible influence on the stretchability for low-noise signal acquisition. 3. The in vitro and in vivo measurements had demonstrated the effect of the metal shielding as well as the photostimulation and recording abilities as a multifunctional biological neural interface.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: