Improvement of the biocompatibility and potential stability of

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Biological and Medical Applications of Materials and Interfaces

Improvement of the biocompatibility and potential stability of chronically implanted electrodes incorporating coating cell membranes Bing Wang, Pengbo Yang, Yaxue Ding, Honglan Qi, Qiang Gao, and Chengxiao Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20542 • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 11, 2019

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ACS Applied Materials & Interfaces

Improvement of the biocompatibility and potential stability of chronically implanted electrodes incorporating coating cell membranes Bing Wang1, Pengbo Yang2, Yaxue Ding1, Honglan Qi1, Qiang Gao1, Chengxiao Zhang1* 1Key

Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education,

School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, 710062, People’s Republic of China 2Department

of Human Anatomy, Histology and Embryology, Health Science Center,

Xi’an Jiaotong University, Xi’an, 710061, People’s Republic of China

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ABSTRACT The development of chronically implanted electrodes attracts much attention since these electrodes are much favorable for monitoring changes of neurotransmitters in brain science. The research of this field mainly was only focused on chemical modification to improve the potential stability, less on the biocompatibility. In this work, at the first time, we proposed the concept of cell-membrane electrodes based on a basic hypothesis using animal’s self-cell membrane to reduce animal exclusiveness (hyperacute rejection and chronic rejection). As a proof of concept, we first studied cell-membrane reference electrodes for chronically implanted electrodes. Red cell membrane (RCM) was extracted from the rat’s blood and coated on the chemically modified Ag/AgCl electrodes. It was found that ionic liquid (IL), 1-butyl-2, 3-dimethylimidazolium hexafluorophosphate (BDMI) showed a good performance rather than Nafion used as coating film for protection of silver chloride on Ag wire and support of the cell-membrane. Electrochemical impedance spectra supported that charge transfer resistance nearly kept constant before and after the electrodes were implanted into the rat’s brain tissues for 28 days. Immunohistochemical analysis of the implant sites in rat’s brain tissues indicated that the extent of glial scarring arising from the Ag/AgCl/BDMI/RCM electrodes was smaller than that of both Ag/AgCl/Nafion electrodes and Ag/AgCl/Nafion/RCM electrodes after 28 days of implantation. The RCM-coated Ag/AgCl/IL electrodes showed a relatively potential stability compared with that of RCM-noncoated Ag/AgCl/IL electrodes after 28 days of implantation. Additionally, the current-voltage curve demonstrated that the RCM-coated electrodes can be used as polarized electrodes. This work demonstrated that the RCM which was coated on the Ag/AgCl/IL electrodes can much improve the biocompatibility and potential stability of the RCM-noncoated Ag/AgCl/IL electrodes 2 / 30


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implanted in rat brain. The cell-membrane coated electrodes will serve as a lighthouse in guiding the design of chronically implanted electrodes for in vivo electrochemical detection. Keywords: cell membrane, implanted electrode, reference electrode, in vivo detection, biocompatibility, brain science

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1. INTRODUCTION The development of electrochemical methods in vivo attracts much attention since electrochemical micro-sensors are much favorable for monitoring changes of neurotransmitters in brain research.1,2 Electrochemical micro-sensors including working electrode and reference electrode for in vivo detection are generally required to be implanted into the brain of animal to obtain electrochemical signals. However, these

implanted

micro-sensors

inevitably

suffer

from

passivation

by

biomacromolecules and proteins while the brain tissues suffer from damage by implanted electrodes.3,4 Therefore, the development of chronically implanted electrodes, which are required to be less damage for the tissues and less passivation for the electrodes, is urgently needed for research in brain science. Some effort has been devoted to the development of implantable electrodes during the past more than 50 years.5 The implantable electrodes generally involve an indicating electrode and a reference electrode for potentiometry, or a working electrode and a reference electrode and an auxiliary electrode for voltammetry and amperometry.6 Since the first device for the rapid and accurate measurement of glucose in intravascular blood using a platinum electrodes modified with enzyme membranes,5 a series of enzyme-based sensors has been reported for glucose in rat peritoneum,7 in rat subcutaneous tissues8 for 10 days, in a skin fold of the neck of dogs.9 However, the tissue surrounding the sensors was cellular, well vascularized.8 Woodward reported that a wire-shape sensor with a diameter of less than 2 mm should evoke minimal reaction in the study of the tissue response to various sensors designed.10 These works reveal that the implantable electrodes/sensors provoke tissues damage and electrode passivation. In recent years, several groups conducted great works on the modification of microelectrodes. Wightman group11, 4 / 30


12

reported a

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series of work on carbon fiber modified with Nafion for in vivo detection of neurotransmitters such as dopamine and 5-hydroxytryptamine in brain research. Recently, Mao group13 reported that a mimic phosphorylcholine polymer film enabled resisting biomolecule adsorption onto the surface of brain-implanted carbon fiber microelectrodes for in vivo monitoring of dopamine. Venton group reported carbon nanotube yarn microelectrodes14 and carbon nanotubes modified metal microelectrodes15 for in vivo detection of dopamine. Gui et al16 reported organic coatings on the electrodes, which limit biofouling by proteins and suffer from sufficiently low impedance. Li et al17

reported cell membrane-mimicking

phosphatidylcholine-terminated

to

monolayers

improve

the

performance

of

electrochemical aptamer-based sensors and to reduce the baseline drift from around 70% to just a few percent after several hours in flowing whole blood in vitro. Additionally, biomimetic membrane modified electrodes have been used as valuable platforms to investigate biologically relevant electroactive molecules embedded in a natural membrane environment and to provide new insights into the mechanism of biological redox cycling.18,

19

All these approaches indicate that suitable modification

on the working electrode can improve the sensitivity and resisting biomolecule adsorption of the implanted microelectrodes. Some effort has been also devoted to improve the potential stability and toxicity of chronically implanted reference electrodes mainly including Ag/AgCl electrodes. In toxic effects of implanted Ag/AgCl reference electrodes. Jackson and Duling20 showed that silver/silver chloride electrodes exerted toxic effects on the smooth muscle and provoked loss of vascular smooth muscle contractility, attributed to the rapid dissolution of silver chloride in the body fluid. In the potential stability of implanted reference electrodes, Velho et al

6

reported that the auxiliary/reference

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potential of both gold and silver-coated sensors presented a cathodic drift, whereas that of Ag/AgCl-coated sensors remained stable only for 1 h. To reduce the toxicity of Ag/AgCl electrode, a series of studies is reported using polymer coatings. Moatti-Sirat et al21 reported that a polyurethane-coated silver/silver chloride reference electrode implanted in rat subcutaneous tissue showed no apparent degradation of the electrode for 10 days, attributed to the fact that a low-permeability polyurethane reduced the dissolution of the silver chloride layer, providing the first evidence that a needle-type glucose sensor miniaturized to a size compatible with clinical use can work for more than 1 week. Moussy et al22 first demonstrated that a cured Nafion-coated Ag/AgCl reference electrode implanted through small skin incisions on the rat neck can significantly improve the potential stability, where the potential shift of cured Nafion-coated Ag/AgCl electrode implanted in rats for 14 days was much smaller than that of Ag/AgCl electrode implanted in rats for 1 week. Hashemi et al23 also reported that a cured Nafion-coated Ag/AgCl reference electrode implanted in the brain of a rat for neurochemical applications and demonstrated that the implanted reference electrode had potential stabile for up to 28 days. Nolan et al24 reported that the potential of Nafion-coated Ag/AgCl electrode can keep stable within 30 - 35 min in water, attributed to the fact that Nafion-coated Ag/AgCl electrode can reduce the leakage of Cl- in aqueous solution. The research of this field mainly was only focused on the coated Ag/AgCl using chemical polymers to improve the potential stability, less on the biocompatibility of the coated Ag/AgCl. Therefore, new materials for improvement of biocompatibility of the coated Ag/AgCl should be explored and assessed. It is well known that cell membrane is made up of a thin layer called the phospholipid bilayer, and negatively charged at physiological pH. He et al25, 6 / 30


26

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developed a new chromatography technique termed as membrane chromatography (CMC) using cell membrane-coated silica particles as separation medium to study drug-receptor interactions and to screen active components from medicinal herbs. This original work suggested that cell membrane can be employed as function materials. In this work, at the first time, we proposed a new concept of cell membrane electrodes based on a basic hypothesis that animal’s self-cell membrane can reduce animal exclusiveness (hyperacute rejection and chronic rejection), should improve the potential stability and biocompatibility of the electrodes in vivo for brain research. As a proof-of-concept, we chosen the a cured Nafion-coated Ag/AgCl reference electrode as a basic electrode and rat’s self-cell membrane was used as a real biologic film. Additionally, two kinds of ionic liquid, 1-butyl-2, 3-dimethylimidazolium hexafluorophosphate (BDMI, cation) and Nafion (anion) used as coating film for protection of silver chloride on silver wire and support of the cell membrane were compared. In this paper, the fabrication of cell membrane implanted Ag/AgCl reference electrodes was presented. The prepared reference electrodes were characterized using scanning electron microscopy and energy dispersive X-ray spectroscopy, electrochemical impedance spectra before and after implantation for 14 days and 28 days. The lesion of implanted reference electrodes in rat brain was evaluated using glial fibrallary acid protein immunoreactivity. The potential stability of the prepared reference electrodes before and after implantation was measured. The work demonstrated that the biocompatibility and potential stability of cell membranes-coated cured IL (Nafion, or BDMI) Ag/AgCl reference electrodes was much improved.

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2. EXPERIMENTAL 2.1 Material and Apparatus 1-Butyl-2, 3-dimethylimidazolium hexafluorophosphate (BDMI), Nafion 117® solution (perfluorinated ionomer, ~5% in a mixture of lower aliphatic alcohols and water), and Triton X-100 were purchased from Sigma-Aldrich (USA). Fibered denture base resin was obtained from Dental Materials Factory of Shanghai Medical Devices Co., Ltd (China). Rabbit Anti-GFAP was obtained from Abcam (UK). Goat Anti-rabbit IgG/RBTIC was obtained from Bioss (China). Pelltobarbitalum natricum was obtained from Xiya Reagent (China). DAB-kit-Pale-brown was obtained from Boster Biological Technology Co. Ltd (China). SP-9001 SP link detection kit (biotin-streptavidin HRP detection systems) was obtained from Zhongshan Goldbridge Biotechnology Co. Ltd (China). Silver wires (0.5 mm o.d.), 2000 mesh sandpapers and 4000 mesh sandpapers were obtained from Gaoss Union Technology Co. Ltd. (China). Neutral balsam, heparin sodium and other reagents with analytical grade were obtained from Sinopharm Chemical Reagent Co., Ltd. (China). Ultrapure water (18.2 MΩ cm, Millipore) was used. 3-18 R desktop high-speed centrifuge (Hunan Hengnuo Instrument Equipment Co. Ltd, China), KQ-250 DE ultrasonic cleaner (Kunshan City Ultrasonic Instrument Co. Ltd, China), CHI 660 E electrochemical workstation (Chenhua, China), stereotaxic frame (RWD Life Science Co. Ltd, China), ZEISS SIGMA field emission scanning electron microscope (SEM) with the energy dispersive X-ray (EDX) spectrometer, FEI Tecnai G2 F20 field transmittance microscope (TEM), CSPM5500 atomic force microscope (AFM, Being Nano-Instruments) were employed in this work. 2.2

Extraction of cell membrane 8 / 30


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The use and care of animals in this study followed the guidelines of the Xi’an Jiaotong University Animal Research Advisory Committee. Female/male Sprague Dawley rats aged 9-12 weeks (about 230-260 g) were obtained from Center for Experimental Animals, Health Science Center, Xi'an Jiaotong University. To get blood, the rats were anesthetized with pelltobarbitalum natricum (40 mg kg-1 rat weight) and then placed in supine position; finally, cardiac blood was drawn out following a classical protocol.27 The red cell membrane (RCM) was extracted from the blood draw out following the procedure reported

28

with some modification.

Briefly, normal saline (4ºC) was added into the drawn-out blood with an approximately equal volume. After the mixture was centrifuged with 3000 r/min for 20 min, the supernatant solution was discarded while the sediment was washed with equal volume of normal saline (4ºC) and centrifuged, repeating this process for 3 times. The resulted sediment was mixed with 20-folds volume of 5 mM Tris-HCl buffer (pH 7.40) in ice-bath and ultrasonically treated on KQ-250 DE ultrasonic cleaner for 30 min (200 W). The treated mixture was centrifuged with 1.1×104 r/min for 20 min and then the sediment was washed with 5 mM Tris-HCl buffer (pH 7.40). The washing and centrifugal procedure repeated until red color (blood cells) in the sediment was invisible. The collected sediment was employed as RCM sample in this work and stored at -20 ºC. The RCM suspension for the work was prepared by suspending a fixed weight of the RCM sample in normal saline (4ºC). The concentration of the RCM suspension was calculated by the weight of the RCM sample and the volume of normal saline. 2.3 Fabrication of RCM-coated Ag/AgCl electrodes The fabrication of RCM-coated Ag/AgCl electrodes was performed in three steps including AgCl coating, ion liquid (IL) coating and RCM coating. Firstly, a silver 9 / 30



wire was treated according to the reference.22 The treated silver wire (3 cm length, 0.5 mm o.d.) was insulated using glue, exposing silver at 0.5 cm in one end for AgCl coating while 0.5 cm in other end for electrical contact. A layer of AgCl on the treated silver wire was formed by a constant current oxidizing with 0.40 mA/cm2 in stirred 0.10 M HCl for 30 min. Secondly, the IL coating was performed by dipping a prepared Ag/AgCl electrode into 1 mL of 0.50 M BDMI (acetone) for 10 s under a slow shake, dried for 30 min. The coating process was continually performed for five times, dried in air at room temperature overnight and then cured at 120 ºC for 1 h. The prepared electrode was termed as Ag/AgCl/BDMI electrode. For the comparison, Ag/AgCl/Nafion electrode was also prepared by the same process using 5% Nafion 117 solution. Finally, after Ag/AgCl/BDMI electrode or Ag/AgCl/Nafion electrode prepared above was sterilized using ultra-violet lamp (15W) for at least 3 h to obtain a sterile surface for the modification of the cell membrane and then dipped into 1 mL of 50 mg/mL RCM prepared with normal saline for 10 s with a slow shake, dried for 10 min, and then recoated for five times, ready for experiment use. The fabricated electrodes were named as Ag/AgCl/BDMI/RCM or Ag/AgCl/Nafion/RCM electrode. 2.4

Implantation of the prepared reference electrodes into the rat brain

Implantation of the prepared reference electrode into the rat brain was performed according to the reference.23 The anesthetized rat was affixed into a stereotaxic frame (RWD Life Science Co. Ltd, China). Flatskull surgical technique was employed by a stereotaxic atlas.29 After a cranial hole on the anesthetized rat was drilled (stereotaxic coordinates relative to bregma: 3.0 mm posterior, 2.5 mm lateral), an electrode prepared as described above was implanted into the thalamus (stereotaxic coordinates relative to bregma: 3.0 mm posterior, 2.5 mm lateral, 5.0 mm ventral). The depth of the electrode implanted into the animal’s brain tissue was 5.0 mm. The implanted 10 / 30


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electrode was stabilized with skull screws and fibered denture base resins. The rat holding the electrode was individually housed in a standard environment on a 12:12 light cycle (lights on at 07:00 am) at a constant temperature, in clear plastic cages with ad libitum access to food and water.30 After 14 days or 28 days of the implantation, the cement surround the reference electrode was softened by carefully dropping a few drops of acetone, and a pinhole on the cement surround the reference electrode was drilled. After that, the reference electrode was removed carefully from the brain tissue and washed with normal saline for further use.

3. RESULTS AND DISCUSSION 3.1 Design and characterization of RCM and fabricated electrodes The new concept of cell membrane electrodes was proposed in this work at the first time. The cell membrane electrodes are defined as the electrodes coated with cell membranes. The basic hypothesis for this concept is original from following fundamentals. Any foreign materials (electrodes, the transplanted organs, agents or organisms, etc.) are harmful to the human body since the human immune system would reject the implanted electrode as a foreign object. If the implanted electrode into an animal was covered with itself cell-membrane, the animal exclusiveness (hyperacute rejection and chronic rejection) should be greatly reduced, so that the biocompatibility and potential stability of the implanted electrode should be greatly improved in vivo for brain research. In the preliminary work, we chosen reference electrode as a model electrode since it is not only widely utilized in potentiometry and voltammetry but also bigger size compared with that of working electrode. Additionally, this idea achieved successfully, it should be extended to the micro-biosensors as indicating/working electrodes. 11 / 30



In the design of Ag/AgCl/IL/RCM electrodes, Ag/AgCl electrode was utilized while BDMI (cation) and Nafion (anion) were chosen as the binder to hold the RCM. Considering that the reference electrodes are implanted into the rat brain, the RCM obtained from left arterial blood of the rat heart was utilized. From the point of animal exclusiveness, the source of the cell membranes is satisfied with the animal itself cell membranes. The reason for choice of red cell membrane (RCM) from cardiac blood of the rats in this work was that it is easily and largely obtained. If this strategy is to be translated clinically, the venous blood can be drawn. For fabrication of Ag/AgCl electrode, it was found that the potential response and stability of the Ag/AgCl electrode prepared using a small constant current method employed by electrochemically oxidizing with 0.40 mA/cm2 in stirred 0.10 M HCl for 30 min were better than that using the cyclic voltammetry reported in the reference,24 attributed to the fact that a dense film of AgCl was formed at a small constant current.

Fig. 1

TEM image (A) of red cell membrane (5 L of 0.5 μg/mL on copper grid, diameter 3

mm) and AFM image (B) of red cell membrane (10 L of 0.5 μg/mL on mica, 10 mm×10 mm).

Characterization of the RCM

The topography of the RCM suspension prepared 12 / 30


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by diluting the RCM suspension (described in 2.2) with normal saline was characterized by TEM and AFM. The TEM image showed that the distribution of the RCM was well-scattered and the average size of the RCM was 80-100 nm (Figure 1, A). The AFM image showed the thickness of the RCM was 3-5mm (Figure 1, B, Fig. S1). This thickness of the RCM is the same as that reported in the reference.31 These observations indicate that the RCM was successfully obtained.

Fig. 2 The representative SEM images (A) and EDX spectra (B) of the fabricated electrodes before

implantation.

(a)

Ag/AgCl;

(b)

Ag/AgCl/Nafion;

Ag/AgCl/Nafion/RCM; (e) Ag/AgCl/BDMI/RCM. 13 / 30


(c)

Ag/AgCl/BDMI;

(d)


Characterization of Ag/AgCl/BDMI/RCM electrodes in preparation

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In order

to check whether the RCM was attached the surface of Ag/AgCl/IL, a series of the electrodes was fabricated as described 2.3, and then characterized using SEM and EDS. Fig.2 shows the representative SEM images (A) and energy dispersive X-ray spectra (B) of the fabricated electrodes before implantation. From Fig.2 (A, a), anomalous particles with a size of about 1 μm are observed, indicating that AgCl is covered on the surface of Ag wire. A crack dense film is observed in Fig. 2 (A, b) and Fig. 2 (A, c), attributed to curing of five films with Nafion or BDMI,22 respectively. Compared Fig. 2 (A, d) with Fig. 2 (A, b) and Fig. 2 (A, e) with Fig. 2 (A, c), more smooth surfaces are observed, indicating that the RCM is coated on the surface of the Nafion film or the BDMI film, respectively. In order to support the successful coatings on Ag wire step by step, EDX spectra was recorded. Fig. 2 (B) shows the X-ray energy spectra of the fabricated electrodes before implantation. The element weight percentages obtained from the peaks in X-ray energy spectra are listed in Table S 1. In the spectrum of the Ag/AgCl electrode (Fig. 2 B, a), four peaks are observed. Two small peaks appear at 0.3 keV for C and 2.25 keV for Au, attributed to the carbon adhesive paste and gold used for enhancing the electroconductivity of the samples in the experiments, and two big peaks appear at 2.8 keV for Cl and at 3 keV for Ag, indicating that AgCl is formed. Compared Fig. 2 (B, b) with Fig. 2 (B, a), two new peaks at 0.52 keV for O and at 0.68 KeV for F (50.34%) appears, ascribed to there exist O and F in Nafion film. Compared Fig. 2 (B, c) with Fig. 2 (B, a), two new peaks at 0.68 keV for F (35.02%) and at 2.03 keV for P (7.49%) appear, ascribed to there exist F and P in BDMI film. Compared Fig. 2 (B, d) with Fig. 2 (B, b), the C element percentage increases from 15.22% to 33.86% and the 14 / 30


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peak of F element disappears, indicating that RCM was coated on the surface of the Ag/AgCl/Nafion electrode. Compared Fig 2 (B, e) with Fig 2 (B, d), the peaks of C were similar and the peak of O increases from 0 to 2.43%, while the peak of F element disappears. And a new peak at 1.05 keV for Na is observed, attributed to the fact that the more amount of Na+ was rejected to the surface of the RCM in the case of existing positively charged BDMI. In summary, the EDX spectra confirmed that the ILs (Nafion or BDMI) and the RCM have been successfully coated on the surface of Ag/AgCl electrode prepared. Additionally, the contact angles on the surfaces of the electrodes (Ag plate) fabricated by the same process described in 2.3 section were measured in triplicate before implantation as showed in Fig. S2 (Supporting Information). The results showed that the contact angle from BDMI (74.5±1.0°) was smaller than that from Nafion (94.1±0.3°), indicating that the wettability of BDMI is better than that of Nafion; while the contact angle from both RCM/BDMI (56.0±3.8°) and RCM/Nafion (59.1±2.7°) were smaller than that from BDMI (74.5±1.0°) and Nafion (94.1±0.3°), respectively. These observations suggest that the hydrophilicity of BDMI is better than that of Nafion, and the RCM coating can improve the hydrophilicity of ion liquids tested. It should be noted that the wettability of a surface is depending on both surface chemistry and surface roughness.32 This issue and both the hydrophobic force and hydrogen bond between the ILs and the RCM should be considered for further study.33 3.2 SEM characterization of the damage of Ag/AgCl/BDMI/RCM electrodes after implantation In order to known the damage of the prepared reference electrodes including Ag/AgCl/BDMI, Ag/AgCl/Nafion, Ag/AgCl/BDMI/RCM and Ag/AgCl/Nafion/RCM 15 / 30



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before and after implantation of the living rat brain were studied using scanning electron microscopy. The SEM images are showed in Fig. 3. All four electrodes studied before implantation showed smooth surfaces. Moussy and Harrison22 reported that a cured Ag/AgCl/Nafion electrode significantly reduce the damage of the non-cured Ag/AgCl/Nafion electrode after implantation. However, they only reported the case up to 14 days of implantation. Therefore, in our work, four electrodes described above were studied up to 28 days of implantation. It was found that the damage of Ag/AgCl/Nafion electrode after 28 days of implantation (Fig. 3, A28) is smaller than that after 14 days of implantation (Fig. 3, A14). This observation is maybe attributed to the fact that the tissue around the implanted electrode was restored since the living animal (rat brain) has a self-repair function.34 Compared the images of Ag/AgCl/BDMI electrodes (Fig. 3, B14, B28) with that of Ag/AgCl/Nafion electrodes (Fig. 3, A14, A28), the film-peeled-off areas on Ag/AgCl/BDMI electrodes (Fig. 3, B14, B28) after both 14 and 28 days of implantation were observed to be relatively smaller than that on Ag/AgCl/Nafion electrodes. These observations indicate that the BDMI film benefits the protection against the damage of the electrode. Compared the film-peeled off areas of the RCM-coated Ag/AgCl/Nafion electrodes (Fig. 3, C14, C28) with that of the Ag/AgCl/Nafion electrodes (Fig. 3, A14, A28), and compared the film-peeled-off areas of the RCM-coated Ag/AgCl/BDMI electrodes (Fig. 3, D14, D28) with that of the Ag/AgCl/BDMI electrodes (Fig. 3, B14, B28), the film-peeled-off areas on the RCM-coated electrodes are smaller than that on the RCM-uncoated electrodes. Additionally, the degree of the damage in the Ag/AgCl/BDMI/RCM

electrode

was

relatively

smaller

than

that

the

Ag/AgCl/Nafion/RCM electrode. In summary, all observations described above demonstrated that BDMI layer is better than Nafion layer for the coating Ag/AgCl 16 / 30


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electrodes and RCM can reduce the damage of the polymer or ion liquid coated Ag/AgCl electrode in subcutaneous implantation in a rat brain.

Fig. 3 The representative SEM images of fabricated reference electrodes. Bar=100 μm. Before implantation,

(A) Ag/AgCl/Nafion;

(B) Ag/AgCl/BDMI;

(C) Ag/AgCl/Nafion/RCM;

(D) Ag/AgCl/BDMI/RCM;

14 days after implantation, (A14) Ag/AgCl/Nafion; (C14) Ag/AgCl/Nafion/RCM; 28 days after implantation, (A28) Ag/AgCl/Nafion; (C28) Ag/AgCl/Nafion/RCM;

3.3

(B14) Ag/AgCl/BDMI; (D14) Ag/AgCl/BDMI/RCM; (B28) Ag/AgCl/BDMI; (D28) Ag/AgCl/BDMI/RCM.

EIS characterization of Ag/AgCl/BDMI/RCM electrodes before and

after implantation The characterization of Ag/AgCl/BDMI/RCM electrodes before and after implantation was also carried out using electrochemical impedance spectroscopy (EIS) in 1.0 mM K3Fe(CN)6–1.0 mM K4Fe(CN)6-0.10 M PBS (pH 7.40) in order to understand the ion permeability of the prepared electrodes. Fig. 4 shows Nyquist plots 17 / 30



of the faradaic impedance spectra of the prepared electrodes before and after implantation and the obtained data are listed in Table S2. The equivalent circuit used for the simulation was presented in our previous work.35 In the equivalent circuit, Rct is the charge transfer resistance; Rs is electrolyte solution resistance; W is mass transferring Warburg impedance; Q is phase angle element; C is double layer capacity. From Fig. 4 (a), the charge transfer resistance (Rct) increases from 19.85 Ω (a) at a bare Ag/AgCl electrode to 123.2 Ω (b) at implanted Ag/AgCl electrode after 14 days, and 147.3 Ω (c) at implanted Ag/AgCl electrode after 28 days, which is the same as reported by Moussy and Harrison.22 The increase of the Rct from bare Ag/AgCl electrode to the implanted Ag/AgCl electrode after 14 days is attributed to the fact that the electrode was surrounded by 1 mm-thick tissue capsule and had been lined with severely inflamed fibrous and granulation tissues after implanting into the tested animals.23 The increase of the Rct from the implanted Ag/AgCl electrode after 14 days to that after 28 days is attributed to the further increase of the tissue capsule. The Rct (33.23 Ω) at Ag/AgCl/BDMI is larger than that (19.85Ω) at Ag/AgCl, attributed to the coating of BDMI film, and smaller than that (100.6 Ω) at the Ag/AgCl/Nafion, attributed to the fact that the BDMI with positive charge makes more easily mass- transfer [Fe(CN)6]3−and [Fe(CN)6]4− to the silver electrode rather than the Nafion with negative charge. More importantly, after implantation for 28 days, the decrease of the Rct value was observed at Ag/AgCl/BDMI/RCM rather than at the Ag/AgCl/Nafion/RCM. This observation can be ascribed to that the tissue capsule surrounding the implanted electrode can increase the Rct while the disruption of the coverage of the coating electrodes can decrease the Rct. The final Rct was depended on the dominated factor of the tissue capsule and the disruption of the coverage. This observation strongly suggests that the RCM coating on 18 / 30


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Ag/AgCl/BDMI electrode is better than that on Ag/AgCl/Nafion. Further discussion will be seen in following section of immunohistochemical analysis.

Fig. 4 Nyquist plots of the impedance spectra of the fabricated electrodes. (A) Ag/AgCl; (B) Ag/AgCl/Nafion; (C) Ag/AgCl/BDMI; (E) Ag/AgCl/Nafion/RCM; (F) Ag/AgCl/BDMI/RCM. The measurements were performed in 1.0 mM K3Fe(CN)6-1.0 mM K4Fe(CN)6-0.10 M PBS (pH 7.40) with the frequency from 100 kHz to 0.1 Hz and 5.0 mV amplitude. The dot line is experiment while the solid line is simulated by an equivalent circuit (D). (a) Black lines were obtained before implantation while (b) red lines and (c) green lines were obtained in 14 days and 28 days after implantation, respectively.

3.4

Immunohistochemical analysis of the implant site in tissue

To gain an insight into the improvement of the biocompatibility of BDMI and RCM coatings to the cortical tissue around the implanted reference electrodes, we carried out immunohistochemical analysis for the cortical tissue nearby the implanted electrode site using a fluorescence image method and 3, 3'-diaminobenzidine (DAB) color image method for glial fibrillary acid protein (GFAP) immunohistochemistry, respectively, described in section 1.4 in Supporting Information. It is well known that GFAP is generally present in normal brain tissue and the number of GFAP increases when brain tissue is damaged. In immunohistochemical method, the immune response 19 / 30



intensity in the images directly corresponds to the density of glial cells, that is, the damage of cortical tissue around the implanted electrode. Therefore, the more number of GFAP observed, the less biocompatibility of the electrodes tested.23 It should be noted the extent of glial scarring also depends on the implanted electrode dimensions. Since the equivalent size of implanted electrodes, their immune response should be comparable.36 Additionally, the intact extent and smooth of cortical tissue around the implanted electrode also corresponds to the damage degree of cortical tissue around the implanted electrode. Fluorescent and DAB staining of GFAP can provide the information of GFAP formed in the tissue surrounding the implanted electrodes and support each other.

Fig. 5 The representative fluorescence images for GFAP immunoreactivity of cortical astroglia in the tissue section stained after the retrieved electrodes. (A14) Ag/AgCl/Nafion, 14 days;

(A28) Ag/AgCl/Nafion, 28 days;

(B14) Ag/AgCl/Nafion/RCM, 14 days;

(B28) Ag/AgCl/Nafion/RCM, 28 days;

(C14) Ag/AgCl/BDMI/ RCM, 14 days;

(C28) Ag/AgCl/BDMI/RCM, 28 days.

Fig. 5 shows the representative fluorescence images of signal antibody bound to 20 / 30


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GFAP surrounding the lesion sites where the electrodes were applied. From Fig. 5, we can see that glial scarring of the tissue section stained is observed, indicating that there exists glial scarring in the lesion where all reference electrodes (A-C)

Fig.6

The representative DAB color images for GFAP immunoreactivity of cortical astroglia in

the tissue section stained after the retrieved electrodes. (A14) Ag/AgCl, 14 days;

(A28) Ag/AgCl, 28 days;

(B14) Ag/AgCl/Nafion, 14days;

(B28) Ag/AgCl/Nafion, 28days;

(C14) Ag/AgCl/BDMI, 14 days;

(C28) Ag/AgCl/BDMI, 28 days;

(D14) Ag/AgCl/Nafion/RCM, 14 days;

(D28) Ag/AgCl/Nafion/RCM, 28 days;

(E14) Ag/AgCl/BDMI/RCM, 14 days;

(E28) Ag/AgCl/BDMI/RCM, 28 days.

21 / 30



Page 22 of 30

were implanted. Furthermore, the fluorescence intensity obtained from Nafion-coated (Fig. 5, A14, A28) is higher than that from Nafion/RCM (Fig. 5, B14, B28) while it obtained from Nafion/RCM (Fig. 5, B14, B28) is higher than that from BDMI/RCM (Fig. 5, C14, C28). These observations suggest that there is small lesion of cortical tissue from BDMI and RCM. Fig 6 shows the representative DAB color images of the GFAP immunoreactivity in cortical astroglia after retrieval of the implanted electrode, representative lesion sites where the different fabricated electrodes were implanted after 14 days or 28 days. In all cases, the implant borders of the lesions at retrieval of Ag/AgCl/Nafion/RCM (Fig.6 D14, D28) and Ag/AgCl/BDMI/RCM (Fig.6 E14, E28) appear smooth, suggesting that the cells are intact. The quantity statistics of glial scarring per selective area (200 m × 200 m) on the image of the GFAP immunoreactivity in the cortical astroglia after retrieval of the implanted reference electrode (Fig. 6) was counted in a double-blind trail to further know that the lesion content of cortical tissue around the implanted reference electrode. The results showed that the counts of glial scarring per selective area (200 μm×200 μm) are 62 cells

for

Ag/AgCl/Nafion,

57

cells

for

Ag/AgCl/BDMI,

38

cells

for

Ag/AgCl/Nafion/RCM, 32 cells for Ag/AgCl/BDMI/RCM, in the cases of 28 days of implantation, respectively. The minimum value of the counts was observed at Ag/AgCl/BDMI/RCM, suggesting that RCM can reduce the lesion of cortical tissue around the implanted electrodes. From this immunohistochemical analysis, the intact extent and smooth of cortical tissues and the relatively lower counts of the glial scarring per selective area in the cases of 28 days of implantation on the image of the GFAP immunoreactivity were observed in the case of Ag/AgCl/BDMI/RCM. These are attributed to the fact that RCM has a good biocompatibility that prevents glial 22 / 30


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cells from adhering to the electrode surfaces. These observations strongly indicate that RCM can reduce the lesion of cortical tissue around the implanted reference electrodes. Additionally, it was found that the count of glial scarring per selective area from Ag/AgCl/BDMI was less than that from Ag/AgCl/Nafion, indicating that the biocompatibility of BDMI is better than that of Nafion. This is possibly ascribed to the fact that BDMI (big cation) has a good protection of silver chloride (with negative charge of the adsorbed chloride) on Ag wire rather than Nafion (big anion). It should note that the cavities observed from the images shown in Fig. 5 and Fig. 6 appeared to suggest size disparity of the implants. This should be ascribed to the differences from tissue tear in the surgical operation for retrieving the implants, rather than from the size of the implants. Study on microglial response and the difference in the extent of lesion should be considered in the further work since it can provide more information of the inconsistent implant size and tissue disruption resulting from the retrieval of the implant. 3.5 Electrochemical evaluation of Ag/AgCl/BDMI/RCM electrodes before and after implantation

Fig. 7

The measured potentials of the fabricated electrodes used as indicating electrodes in 0.10

M KCl.

23 / 30



The potentials of the reference electrodes fabricated were evaluated before and after implantation using an open circuit potential-time technology. In this evaluation, the fabricated electrode was used as an indicating electrode while a commercial saturated calomel electrode was used as a reference electrode. These two electrodes were immersed in 0.10 M KCl with a salt bridge separating the half-cells. The obtained potentials of the reference electrodes are showed in Fig. 7, and the obtained data are listed in Table S3. Before implantation of the fabricated reference electrodes, the small variation tendency of the potentials at all tested electrodes are observed (Fig 7, black columns). This variation tendency is similar with that reported.22 After implantation of the fabricated electrodes for 14 days, the order of the negatively shifted potential is: Ag/AgCl > Ag/AgCl/Nafion/RCM = Ag/AgCl/BDMI/RCM > Ag/AgCl/Nafion > Ag/AgCl/BDMI (Fig 7, red columns). After implantation of the fabricated electrodes for 28 days, the order of the negatively shifted potential is: Ag/AgCl > Ag/AgCl/Nafion > Ag/AgCl/BDMI > Ag/AgCl/Nafion/RCM > Ag/AgCl/BDMI/RCM (Fig. 7, blue columns). The results indicate that ILs can prevent from abscission of AgCl. Additionally, the potential stability of the electrodes containing BDMI was better than that containing Nafion. This is attributed to the fact that the mass transport of chloride in positively charged BDMI film is bigger than that in negatively charged Nafion film since chloride was the response ion for Ag/AgCl electrode. Importantly, RCM-coated electrodes showed relatively small changes on the potentials. This could be attributed to the fact that the RCM contains ion channels which benefit mass transfer of chloride, and indicates that RCM is good modification materials for the preparation of the Ag/AgCl electrodes. In order to know that the possible applications of the fabricated electrode to working electrode, cyclic voltammograms were obtained at a classical three system 24 / 30


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consisting of the fabricated electrode as a working electrode, a large Pt as an auxiliary electrode and a commercial Ag/AgCl (Sat. KCl) as a reference electrode in 0.10 M KNO3 with a scan rate of 100 mV/s in the potential range from 0 V to -0.2 V (vs. Sat. KCl) (Fig. S3). The cyclic voltammograms showed that before implantation, the absolute

value

of

Ag/AgCl/BDMI/RCM

the at

cathodic

peak

current

-0.2

was

bigger

V

(-2.15 than

mA)

that

obtained

(-1.9

mA)

on on

Ag/AgCl/Nafion/RCM at -0.2 V, while after 28 days of implantation, the absolute value of the cathodic peak current (-1.6 mA) obtained on Ag/AgCl/BDMI/RCM at -0.2 V was bigger than that (-0.78 mA) obtained on Ag/AgCl/Nafion/RCM at -0.2 V. This observation indicates that the RCM-coated electrodes could be used as a polarized electrode and possible application as the polarized electrode in two-electrode system for glucose microsensors.

4. Conclusion In this work, we proposed a new idea of cell-membrane electrodes for implantation into animals for in vivo detection at the first time based on a basic hypothesis that animal's cell-membrane which is coated on the surface of the implanted electrode can reduce the animal exclusiveness, and the biocompatibility and potential stability of the implanted electrode should be greatly improved for brain research in vivo. In preliminary work, we studied the cell membrane reference electrodes. It was found that BDMI is better Nafion as a coated-material for Ag/AgCl electrode, attributed to the positive charge of BDMI. RCM-coated Ag/AgCl/IL electrodes showed a relatively less damage of the rat brain tissues compared with that of RCM-noncoated Ag/AgCl/IL electrodes from SEM and immunohistochemical analysis after 28 days of implantation. Potential measurements results demonstrated 25 / 30



Page 26 of 30

that RCM-coated Ag/AgCl/IL electrodes showed a relatively potential stability compared with that of RCM-noncoated Ag/AgCl/IL electrodes. Additionally, the result demonstrated that the RCM-coated electrodes can be used as a polarized electrode. This work demonstrated that the rat’s RCM-coated on Ag/AgCl/BDMI electrodes can much improve the biocompatibility and potential stability in rat brain compared with cured Nafion-coated Ag/AgCl electrode up to 28 days of implantation. The further work on a comparison of RCM coating with the cell-membrane-mimic chemical

coating

such

as

phosphorylcholine

polymer,

lipid

bilayers,

or

phosphatidylcholine-terminated SAMs should be considered to demonstrate the advantageous of the RCM coating. Additionally, electrochemical evaluation using the retrieved electrodes could be obscured by the extraction process from the brain tissue. It would be more informative if such study were carried out in vivo, similar to chronic recording with implanted neural electrodes. The cell membrane-coated electrode will serve as a lighthouse in guiding the design of chronically implanted electrodes for in vivo electrochemical detection. The study on cell membrane oxygen electrode and cell membrane pH electrode in living animals is going on in our Lab. ASSOCIATED CONTENT Supporting Information Experimental details of the fabrication of Ag/AgCl electrode and Ag/AgCl/IL electrodes, electrochemical measurements, immunohistochemistry for GFAP; Figures of AFM image of RCM and profile analysis for the AFM image, contact angle images of different fabricated electrodes, and cyclic voltammograms; Tables of EDX weight percentage different fabricated electrodes, the charge transfer resistance of different electrodes and the measured potentials of the fabricated reference electrodes before and after implantations. 26 / 30


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS The funding of the National Science Foundation of China (No. 91332101) and the Fundamental Research Funds for the Central Universities (No. 2016TS048) are gratefully acknowledged.

REFERENCES 1 Robinson, D. L.; Herman, A.; Seipel, A. T.; Wightman, R. M. Monitoring Rapid Chemical Communication in the Brain. Chem. Rev. 2008, 108, 2554-2584. 2 Zhang, M.; Yu, P.; Mao, L. Rational Design of Surface/Interface Chemistry for Quantitative in Vivo Monitoring of Brain Chemistry. Accounts Chem. Res. 2012, 45, 533-543. 3 Wilson, G. S.; Johnson, M. A. In-Vivo Electrochemistry: What Can We Learn about Living Systems? Chem. Rev. 2008, 108, 2462-2481. 4 Soto, R. J.; Hall, J. R.; Brown, M. D.; Taylor, J. B.; Schoenfisch, M. H. In Vivo Chemical Sensors: Role of Biocompatibility on Performance and Utility. Anal. Chem. 2017, 89, 276-299. 5 Clark, L. C. Jr.; Lyons, C. Electrode Systems for Continuous Monitoring in Cardiovascular Surgery. Ann. N.Y. Acad. Sci. 1962, 102, 29-45. 6 Velho, G.; Froguel, P.; Sternberg, R.; Thevenot, D. R.; Reach, G. In Vitro and In Vivo Stability of Electrode Potentials in Needle-Type Glucose Sensors. Diabetes 1989, 38, 164-171. 7 Clark, L. C. Jr.; Noyes, L. K.; Spokane, R. B.; Sudan, R.; Miller, M. L. Long-Term Implantation of 27 / 30



Voltammetric Oxidase/Peroxide Glucose Sensors in the Rat Peritoneum. Methods Enzymol. 1988, 137, 68-89. 8 Ertefai, S.; Gough, D. A. Physiological Preparation for Studying the Response of Subcutaneously Implanted Glucose and Oxygen Sensors. Med. Eng. Phys. 1989, 11, 362-368. 9 Moussy, F.; Harrison, D. J.; O’Brien, D. W.; Rajottet, R. V. Performance of Subcutaneously Implanted Needle-Type Glucose Sensors Employing a Novel Trilayer Coating. Anal. Chem. 1993, 65, 2072-2077. 10 Woodward, S. C. How Fibroblasts and Giant Cells Encapsulate Implants: Considerations in Design of Glucose Sensors. Diabetes Care 1982, 5, 278-281. 11 Hashemi, P.; Dankoski, Elyse C.; Petrovic, J.; Keithley, R. B.; Wightman, R. M.; Voltammetric Detection of 5-Hydroxytryptamine Release in the Rat Brain. Anal. Chem. 2009, 81, 9462-9471. 12 Keithley, R. B.; Takmakov, P.; Bucher, E. S.; Belle, A. M.; Owesson-White, C. A.; Park, J.; Wightman, R. M. Higher Sensitivity Dopamine Measurements with Faster-Scan Cyclic Voltammetry. Anal. Chem. 2011, 83, 3563-3571. 13 Liu, X.; Xiao, T.; Wu, F.; Shen, M. Y.; Zhang, M.; Yu, H.; Mao, L. Ultrathin Cell-Membrane-Mimic Phosphorylcholine Polymer Film Coating Enables Large Improvement for in Vivo Electrochemical Detection. Angew. Chem. Int. Ed. 2017, 56, 11802-11806. 14 Yang, C.; Trikantzopoulos, E.; Nguyen M. D.; Jacobs C. B.; Wang, Y.; Mahjouri-Samani, M.; Ivanov, I. N.; Venton, B. J. Laser Treated Carbon Nanotube Yarn Microelectrodes for Rapid and Sensitive Detection of Dopamine in Vivo. ACS Sens. 2016, 1, 508-515. 15 Yang, C.; Jacobs, C. B.; Nguyen, M. D.; Ganesana, M.; Zestos, A. G.; Ivanov, Ilia N.; Puretzky, A. A.; Rouleau, C. M.; Geohegan, D. B.; Venton, B. J. Carbon Nanotubes Grown on Metal Microelectrodes for the Detection of Dopamine. Anal. Chem. 2016, 88, 645-652. 16 Gui, A. L.; Luais, E.; Peterson, J. R.; Gooding, J. J. Zwitterionic Phenyl Layers: Finally, Stable, Anti-Biofouling Coatings that Do Not Passivate Electrodes. ACS Appl. Mater. Interfaces 2013, 5, 4827-4835. 17 Li, H.; Dauphin-Ducharme, P.; Arroyo-Curras, N.; Tran, C. H.; Vieira, P. A.; Li, S.; Shin, C.; Somerson, J.; Kippin, T. E.; Plaxco, K. W. A Biomimetic Phosphatidylcholine-Terminated Monolayer Greatly Improves the in Vivo Performance of Electrochemical Aptamer-Based Sensors. Angew. Chem. Int. Ed. 2017, 56, 7492-7495. 18 Ma, W; Li, D. W.; Sutherland, T. C.; Li, Y.; Long, Y. T.; Chen, H. Y. Reversible Redox of NADH and NAD+ at a Hybrid Lipid Bilayer Membrane Using Ubiquinone, J. Am. Chem. Soc. 2011, 133, 12366-12369 19 Ma, W.; Ying, Y. L.; Qin, L. X.; Gu, Z.; Zhou, H.; Li, D. W.; Sutherland, T. C.; Chen, H. Y.; Long, Y. T. Investigating Electron-Transfer Processes Using a Biomimetic Hybrid Bilayer Membrane System, Nat. Protoc. 2013, 8, 439-450. 20 Jackson, W. F.; Duling, B. R.; Toxic Effects of Silver-Silver Chloride Electrodes on Vascular Smooth Muscle. Circ. Res. 1983, 105-108. 28 / 30


Page 28 of 30

Page 29 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


21 Moatti-Sirat, D.; Capron, F.; Poitout, V.; Reach, G.; Bindra, D. S.; Zhang, Y.; Wilson, G. S.; Thevenot, D. R. Towards Continuous Glucose Monitoring: In Vivo Evaluation of A Miniaturized Glucose Sensor Implanted for Several Days in Rat Subcutaneous Tissue. Diabetologia 1992, 35, 224-230. 22 Moussy, F.; Harrison, D. J. Prevention of The Rapid Degradation of Subcutaneously Implanted Ag/Agcl Reference Electrodes Using Polymer Coatings. Anal. Chem. 1994, 66, 674-679. 23 Hashemi, P.; Walsh, P. L.; Guillot, T. S.; Gras-Najjar, J.; Takmakov, P.; Crews, F. T.; Wightman, R. M. Chronically Implanted, Nafion-Coated Ag/AgCl Reference Electrodes for Neurochemical Applications. ACS Chem. Neurosci. 2011, 2, 658-666. 24 Nolan, M. A.; Tan, S. H.; Kounaves, S. P. Fabrication and Characterization of a Solid State Reference Electrode for Electroanalysis of Natural Waters with Ultramicroelectrodes. Anal. Chem. 1997, 69, 1244-1247. 25 He, L.; Geng, X. Cell Membrane Chromatography-a New Method for Studying the Drug-Receptor Interactions. New Progress for Biomed. Chromatogr. 1996, 3, 8-9. 26 He, L.; Wang, S.; Yang, G.; Zhang, Y.; Wang, C.; Yuan, B.; Hou, X. Progress in Cell Membrane Chromatography. Drug Discov. Ther. 2007, 1, 104-107. 27 Joslin, J. O. Blood Collection Techniques in Exotic Small Mammals. J. Exot. Pet Med. 2009, 18, 117-139. 28 Du, H.; Ren, J.; Wang, S.; He, L. Cell Membrane Chromatography Competitive Binding Analysis for Characterization of α1A Adrenoreceptor Binding Interactions. Anal. Bioanal. Chem. 2011, 400, 3625-3633. 29 Paxinos, G.; Watson, C. The Rat Brain in Stereotaxic Coordinates, fifth edition; Academic Press, 2005. 30 Xiang, L.; Yu, P.; Zhang, M.; Hao, J.; Wang, Y.; Zhu, L.; Dai, L.; Mao, L. Platinized Aligned Carbon Nanotube-Sheathed Carbon Fiber Microelectrodes for in Vivo Amperometric Monitoring of Oxygen. Anal. Chem. 2014, 86, 5017-5023. 31 Andersen, O. S.; Koeppe, R. E. II. Bilayer Thickness and Membrane Protein Function: An Energetic Perspective. Annu. Rev. Biophys. Biomol. Struct. 2007, 36, 107-130. 32 Boduroglu,S.; Cetinkaya,M.; Dressick,W. J.; Singh, A.; Demirel, M. C. Controlling the Wettability and Adhesion of Nanostructured Poly-(p-xylylene) Films, Langmuir 2007, 23, 11391-11395 33 Wang, Y. Q.; Li, M. Y.; Qiu, H.; Cao, C.; Wang, M. B.; Wu, X. Y.; Huang, J.; Ying, Y. L.; Long, Y. T. Identification of Essential Sensitive Regions of the Aerolysin Nanopore for Single Oligonucleotide Analysis, Anal. Chem. 2018, 90, 7790−7794. 34 Zhang, Y.; Strehin, I.; Bedelbaeva, K.; Gourevitch, D.; Clark, L.; Leferovich, J.; Messersmith, P. B.; Heber-Katz, E. Drug-Induced Regeneration in Adult Mice. Sci Transl Med. 2015, 290, 290ra292, DOI:10.1126/scitranslmed.3010228. 35 Wang, B.; Jing, R.; Qi, H.; Gao, Q.; Zhang, C. Label-Free Electrochemical Impedance Peptide-Based Biosensor for the Detection of Cardiac Troponin I Incorporating Gold 29 / 30



Nanoparticles Modified Carbon Electrode. J. Electroanal. Chem. 2016, 781, 212-217. 36 Jaquins-Gerstl, A.; Michael, A. C. Comparison of the Brain Penetration Injury Associated with Microdialysis and Voltammetry. J. Neurosci. Methods 2009, 183, 127-135.

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Improvement of the biocompatibility and potential stability of

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