Electrophoretically Sheathed Carbon Fiber Microelectrodes with Metal

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Electrophoretically Sheathed Carbon Fiber Microelectrodes with Metal/Nitrogen/Carbon Electrocatalyst for Electrochemical Monitoring of Oxygen In Vivo Yang Cao, Wenjie Ma, Wenliang Ji, Ping Yu, Fei Wu, Huixia Wu, and Lanqun Mao ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00100 • Publication Date (Web): 12 Feb 2019 Downloaded from http://pubs.acs.org on February 17, 2019

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ACS Applied Bio Materials

Electrophoretically

Sheathed

Carbon

Fiber

Microelectrodes

with

Metal/Nitrogen/Carbon

Electrocatalyst for Electrochemical Monitoring of Oxygen In Vivo Yang Cao,†, § Wenjie Ma,‡, ξ, § Wenliang Ji,‡ Ping Yu,‡, ξ Fei Wu,‡, ξ Huixia Wu,*, † and Lanqun Mao*, ‡, ξ † The

Key Laboratory of Resource Chemistry of Ministry of Education, Shanghai Key Laboratory of Rare Earth

Functional Materials, Shanghai Municipal Education Committee Key Laboratory of Molecular Imaging Probes and Sensors, Shanghai Normal University, Shanghai 200234, China ‡

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living

Biosystems, Institute of Chemistry, The Chinese Academy of Sciences (CAS), CAS Research/Education Center for Excellence in Molecule Science, Beijing 100190, China ξ University

of Chinese Academy of Sciences, Beijing 100049, China.

Keywords: in vivo analysis; M/N/C nanomaterials; O2 detection; metal-organic framework; oxygen reduction reaction.

§ These

authors contributed equally. 1

ACS Paragon Plus Environment

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*Corresponding

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Authors. E-mails: [email protected], [email protected], Fax: +86-10-62559373.

ABSTRACT Metal/nitrogen/carbon (M/N/C, M = Co and/or Fe) nanomaterials have been demonstrated to catalyze oxygen reduction reaction (ORR) with implication in fuel cell studies, however, their potential application as sensing materials for in vivo monitoring of oxygen (O2) in the central nervous system has never been reported. This study reports a first demonstration on that M/N/C nanomaterials can be used as sensing materials to form an electrochemical assay for in vivo O2 monitoring. To demonstrate this application, the M/N/C nanocomposites prepared by pyrolysis of zeolitic imidazolate framework (ZIF-67) is used as an example and is electrophoretically deposited onto carbon fiber microelectrodes (CFEs) to catalyze a four-electron reduction of O2 without producing cell-toxic hydrogen peroxide intermediate. The M/N/C-sheathed CFEs have high catalytic performance toward ORR in a neutral solution, selectivity toward O2 sensing in the presence of ascorbic acid (AA), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), uric acid (UA), and 5-hydroxytryptamine (5HT) at their physiological levels in rat brain, and capability to real-time monitor O2 fluctuation during respiring gases. This study offers a new electrochemical approach to in vivo O2 monitoring with non-platinum catalyst for ORR.

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INTRODUCTION In recent years, non-noble metal incorporated carbon nanomaterials have been developed as electrocatalysts for a variety of reactions such as oxygen reduction reaction (ORR),1-3 hydrogen evolution reaction (HER),4 oxygen evolution reaction (OER),5 and hydrocarbon conversion reactions for fuel cells.6 Typically, M/N/C (M = Co and/or Fe) nanomaterials synthesized by pyrolyzing precursors consisting of transition metals, nitrogen, and carbon have been recognized to be one of the most promising carbon-based ORR electrocatalysts owing to their high activity, high stability, parasitic active sites and simple yet efficient synthesis process.1 To date, concentrated researches have been devoted to the synthesis of different precursors, development of various strateges for the M/N/C preparation, as well as the structure/property study on the catalysts to make them situable for energy-related applications. Nevertheless, there has been no report on the application of functional M/N/C nanomaterials for in vivo analysis so far, although such electrocatalysts have great potential in electroanalysis due to their outstanding features including simple fabrication, high activity, and high stability. As well demonstrated previously, O2 participates in many biochemical reactions and plays a critical role in an extensive range of physiological and pathological processes.7 For instance, O2 is an important indicator of neuronal activity in neurochemical processes; abnormal fluctation of O2 in brain interferes with the neurochemical processes and even induces neural dysfunction.8-9 Typically, elevated concentrations of O2 causes cerebral oxygen toxicity, while oxygen deficiency is connected to pathological conditions like brain ischemia.10 It was reported that global brain ischemia caused a range of neurochemical changes and eventually led to long-term histologicaland functional damage, in which O2 plays key roles.11 Therefore, a straightforward approach to providing information on the dynamics of O2 fluctuation in the brain in a real-time nature would be useful to understand the physiological and pathological roles of O2. Up to now, some methods have been deveopled based on the intrinsinc physical and chemical properties of O2 for in vivo determination of O2, such as biopsy technique, Raman spectroscopy, near-infrared spectroscopy, 3



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fluorescence or phosphorescence spectroscopy, nuclear magnetic resonance, and electron spin resonance.12-14 However, these methods are normally confronted with several limitations such as complicated instrumentation and restrained detection depth, making in vivo monitoring of O2 very challenging. Compared with these methods, in vivo voltammetry employing microelectrodes directly implanted into the brain regions holds a promising position in the study of physiology and pathology because of the advantages in high spatiotemporal resolution and capacity for real-time and in situ detection.15-16 The earliest use of in vivo voltammetry for O2 detection can be traced back to the year 1958.17 Clark and his colleagues successfully measured the O2 level of animal brain tissue and myocardial using platinum or pyrolytic graphite encapsulated electrodes. In subsequent studies, platinum or gold microelectrode, platinum-plated carbon ring electrode were employed to determine the O2 concentration in cells or biological tissues and real-time monitor the dynamics of O2 under the physiological and pathological conditions of electrical irritation, neurotransmitter release, ischemia, hyperoxia, anesthesia and injection of various drugs.18-22 Although Pt facilitates four-electron ORR with high activity, one of the most challenging problems arises from the poor stability of Pt under the complicated physiological environment. The platinum-based electrode is susceptible to surface poisoning by contaminants such as chloride ions and thus protective and gas-selective membranes are essentially required. Besides, the platinum electrocatalysts also may lose stability due to electrochemical dissolving, agglomerating, and detaching.23-24 In order to suppress Pt dissolution and detachment during electrochemical reduction, Pt nanoparticles are homogeneously distributed onto a support. In a previous study, our group developed an electrochemical method by platinizing carbon nanotubes aligned onto CFEs for in vivo monitoring of O2.25 In spite of good analytical properties of the electrodes, the complicated fabrication procedure however provides a barrier to its wide in vivo application. Compared with Pt-based materials, carbon-based materials are advantageous because they are highly resistant to surface poisoning26-27 and are thus able to serve as electrode materials for continuously running the measurements over extended periods. However, reduction of O2 at carbon-based electrodes normally undergoes a two-electron process, generating toxic intermediate H2O2. Although oxygen reduction reaction (ORR) activities can be enhanced by using electrocatalysts such as hemin, laccase and porphyrin adsorbed onto carbon nanomaterials,28-30 the stability for the electrocatalysis needs to be further improved. Therefore, searching for 4


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new carbon-based electrocatalysts for ORR with a high efficiency and stability has been a longstanding focus in not only energy conversion technologies but also in vivo O2 sensing. In this article, we report a first demonstration to use Co/N/C electrocatalyst to develop a new method for in vivo O2 sensing by electrophoretically depositing the catalyst onto carbon fiber microelectrodes (Scheme 1). The Co/N/C electrocatalyst with parasitic active catalytic sites synthesized by pyrolyzing ZIF-67 possesses high catalytic performance towards ORR and, as a consequence, the as-fabricated microelectrode responds well to O2 sensitively and selectively in rat hippocampus. This study offers an effective platform for in vivo O2 monitoring, which enables the studies on various physiological and pathological processes related to brain O2.

Scheme 1. Illustration of the application of Co/N/C catalysts for in vivo monitoring of O2. (1) Co/N/C catalysts were obtained by pyrolyzing ZIF-67 at 700 °C. (2) The sensor was fabricated by electrophoretically depositing the catalyst onto carbon fiber microelectrode. (3) The as-fabricated sensor was applied for in vivo monitoring of O2.

EXPERIMENTAL SECTION Reagents and Solutions. Co(NO3)2·6H2O, 2-methylimidazole, methanol, dopamine (DA), 3,4dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), uric acid (UA),

Nafion (5%), and 5-

hydroxytryptamine (5-HT) were purchased from Sigma and used without further purification. Artificial cerebrospinal fluid (aCSF) was prepared by mixing KCl (2.4 mM), NaCl (126 mM), MgCl2 (0.85 mM), CaCl2 (1.1 mM), KH2PO4 (0.5 mM), NaHCO3 (27.5 mM), and Na2SO4 (0.5 mM) in water and the solution pH was adjusted to 7.4. All other chemicals were at least analytical reagents and used as supplied. Aqueous solutions 5



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were all prepared with pure water obtained from a Milli-Q water system (Millipore, 18.2 MΩ cm). Unless mentioned otherwise, all experiments were conducted at room temperature. Synthesis of Co/N/C Catalysts. Co/N/C catalysts were synthesized with pyrolysis of ZIF-67. ZIF-67 was prepared as described previously with slight change in the procedure.31 In a typical experiment, two methanolic solutions were prepared by dissolving 1.436 g of Co(NO3)2·6H2O and 3.244 g of 2-methylimidazole into 100 mL of methanol, and then the two solutions were mixed under vigorous stirring. The resulting mixture was constantly stirred overnight. The obtained solid was collected by centrifugation and washing with methanol repeatedly for several times, followed by vacuum drying at 150 °C for 8 h. The prepared ZIF-67 precursor was pyrolyzed on quartz tubes in an electric furnace under nitrogen atmosphere at different temperatures of 600, 700, 800, and 900 °C for 2 h and cooled down naturally to room temperature. The pyrolysis products of ZIF-67 at different temperatures were named as ZIF-67-600, ZIF-67700, ZIF-67-800, and ZIF-67-900. Preparation of ZIF-67-700-Modified-Carbon Fiber Microelectrodes (ZIF-67-700-CFEs). Fabrication and treatment of CFEs was performed following the procedures reported previously.11 The exposed carbon fibers were shortened into a length of 300-500 μm using a surgery scalpel under a microscope. The obtained CFEs were washed by sonication in acetone, 3 M HNO3, 1.0 M KOH, and pure water sequentially, each for 23 min. Subsequently, the electrodes were subjected to electrochemical pre-activation with chronopotentiometry by keeping the potential at +1.5 V for 80 s, and then with cyclic voltammetry by scanning the electrodes within 0 to 1.0 V with a scan rate of 0.05 V s-1 in 1.0 M KOH until a stable cyclic voltammogram was obtained. The ZIF-67-700 catalyst was confined onto the surface of CFEs by electrophoresis deposition in an aqueous dispersion of the catalyst (2 mg mL-1) with chronoamperometry at -1.0 V for different times. The electrodes were taken out of the dispersion, rinsed with pure water, and dried at room temperature. Apparatus and Measurements. The morphology of ZIF-67 precursor, Co/N/C catalyst, CFEs and ZIF67-700-CFEs were characterized by scanning electron microscope (S-4800, Hitachi, Japan). Transmission electron microscopy (TEM) images were acquired by JEOL JEM-2011F (100 kV). Powder X-ray diffraction (PXRD) patterns were obtained with an X-ray diffractometer using 18 kW Cu-Kα radiation (D/max 2500, Rigaku, Japan). Thermal gravimetric analysis (TGA) was carried out on a thermal analyzer (STA 409 PC/PG, 6


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Netzsch, Germany). X-ray photoelectron spectroscopy (XPS) was performed using an electron spectrometer with 300 W Al-Kα radiation (ESCALab220i-XL, VG Scientific, America). The ZIF-67-700-CFEs prepared at different electrophoretic deposition time were also characterized by a scientific grade metallographic microscope 10XB-P (Shanghai, China). Rotating ring-disk electrode (RRDE) voltammetric curves were acquired on an electrochemistry workstation (WaveDrive10, Pine Instruments), with Pt wire as counter electrode and Ag/AgCl (KCl, 3 M) electrode as reference electrode. For the preparation of working electrodes, 4 mg of Co/N/C catalyst prepared at various temperatures was dispersed in 2 mL of pure water with constant sonication for 1 h. Then, 50 μL of the suspension was drop-casted onto glassy carbon disk electrode with the diameter of 5 mm. The modified electrode was dried under ambient conditions, followed by dropwise coating of 0.5% Nafion (5 μL). RRDE voltammograms were obtained by scanning the disk electrode in a cathodic direction at 10 mV s-1, while keeping the potential of the ring electrode constant at +0.5 V for the oxidation of hydrogen peroxide (H2O2). The aCSF used as electrolyte was bubbled with O2 for 20 min, and the measurements were performed under the O2 atmosphere. Other electrochemical measurements were conducted with a computer-controlled electrochemical analyzer (CHI 730D, Chenhua, China). ZIF-67-700-CFEs and a micro-sized platinum wire were used as working electrode and counter electrode, respectively. A tissue-implantable microsized Ag/AgCl (aCSF) electrode, prepared according to previous report,32 was used as reference electrode. Calibration of the ZIF-67-700-CFEs was carried out by measuring the O2 reduction current in aCSF saturated with N2, ambient air, and O2 with the O2 concentrations of 0, 200, and 1250 μM, respectively.25 In Vivo Experiments. Adult male Sprague-Dawley rats (300-350 g) were obtained from Health Science Center (Peking University). The rats were kept on a 12:12 h light-dark schedule and fed with food and water ad libitum. The use and care of rats were approved and guided by the Institutional Animal Care and Use Committee of National Center for Nanoscience and Technology of China. The rats were anesthetized with isoflurane (4 % induction, 2 % maintenance) through a R520 gas pump (RWD, China) and positioned onto a stereotaxic apparatus via the ear rods during in vivo experiments. After the brain region was confirmed accurately, the ZIF67-700-CFEs were implanted into the hippocampus (AP = 5 mm, L = 5 mm from bregma, V = 4.5 mm from 7



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dura) using standard stereotaxic procedures. Respiring gas experiments were conducted by exposing the animal to O2 for 120 s, N2 for 40 s, or CO2 for 150 s. The gases were stored in rubber bladders and delivered through a tube that was positioned closely to the nose of the rats. In vivo measurements of O2 in rat brain was conducted amperometrically with the ZIF-67-700-CFEs at a constant potential of -0.5 V. RESULTS AND DISCUSSION Characterization of ZIF-67 and Co/N/C Catalyst. Metal organic frameworks have emerged as a new platform for preparing highly efficient ORR electrocatalysts containing M/N/C nanomaterials, among which ZIF-67 has been demonstrated to be an excellent precursor because of the existence of abundant cobalt and nitrogen species.33 In the present study, ZIF-67 was synthesized and characterized by TGA, PXRD, SEM and XPS. As displayed in TGA curve (Figure 1 A), ZIF-67 only shows ~6% of weight loss up to a temperature of 500 °C due to the removal of guest molecules, suggesting high thermal stability of ZIF-67. The weight of the sample decreases abruptly at 500-600 °C, because of the decomposition of the framework structure to form Co and CoxOy nanoparticles decorated graphitic carbon materials. Based on the results of TGA analysis, the temperature for pyrolyzing ZIF-67 was chosen to be higher than 600 °C. Considering the pyrolysis temperature had a great influence on the crystallinity and composition of the products (i.e., Co/N/C), four samples were prepared by pyrolyzing ZIF-67 precursor at 600, 700, 800, and 900 °C in nitrogen atmosphere. The PXRD patterns of ZIF-67 and Co/N/C samples were displayed in Figure 1 B. All observed diffraction peaks for ZIF67 matched well with the reported data.31, 33 For ZIF-67-900, diffraction peaks at around 44.3°, 51.5° and 75.9° were observed, which were assigned to the cubic structure of Co nanoparticles (PDF#15-0806). The other three Co/N/C samples obtained at the pyrolysis temperature lower than 900 °C (i.e., ZIF-67-600, ZIF-67-700, and ZIF-67-800) exhibit several new diffraction peaks in addition to those of Co nanoparticles. The peaks at 31.8°, 36.6°, 59.4° and 65.5° were attributed to the crystal planes of Co3O4 nanoparticles (PDF#43-1003), the peaks at 37.3°, 62.1° and 77.7° were from CoO nanoparticles (PDF#43-1004). The broad peak at ~24° in the patterns of four Co/N/C samples demonstrated the formation of a well-developed graphitic structure with high content of sp2 carbon. From these results, we could draw a conclusion that higher pyrolysis temperature (> 900 °C) resulted in the formation of elemental cobalt, while relatively lower temperature promoted the formation of cobalt oxide. During the pyrolysis, ZIF-67 would gradually decompose accompanied with the conversion of Co2+ into metallic 8


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Co. At relatively lower temperature, the Co would be oxidized to cobalt oxide by oxygen-containing species. At higher pyrolysis temperature, cobalt oxide phase would be further reduced by the graphitic carbon to Co.34 Given that ZIF-67-700 exhibits the best electrocatalytic performance for ORR (as demonstrated below), consequent studies were carried out to achieve more information on the morphology and composition of ZIF67-700. As could be seen from the SEM images (Figure 1 C and D), ZIF-67 crystals show relatively uniform size (150 ± 22 nm) and well-defined clear boundaries, while ZIF-67-700 has a loose appearance with no obvious boundary, which is good for O2 sensing since the porous structure of ZIF-67-700 would facilitate the diffusion of O2 during electrocatalysis. TEM images and EDX elemental mappings show that N, O, and cobalt were homogenously distributed over the carbon skeleton (Figure 1 E and F).

Figure 1. (A) TGA curve of ZIF-67. (B) PXRD patterns of ZIF-67 (black), ZIF-67-600 (red), ZIF-67-700 (blue), ZIF-67-800 (pink) and ZIF-67-900 (green). (C) SEM image of ZIF-67. Inset, size distribution of ZIF-67. (D) SEM image of Co/N/C catalyst (ZIF-67-700). (E) TEM image of Co/N/C catalysts (ZIF-67-700). (F) EDX elemental mappings of a Co/N/C particle; C (red), N (yellow), O (green) and Co (blue). Scale bar, 50 nm.

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Figure 2 displays high resolution N1s and Co2p XPS spectra of ZIF-67 and ZIF-67-700 samples. The N1s signal of the sample shifts to a higher binding energy area (Figure 2 A and C), indicating the as-formed ZIF67-700 was highly graphitized. The N1s peaks of ZIF-67-700 (Figure 2 C) were deconvoluted into the components of pyridinic N, Co-Nx bonding, pyrrolic N and graphitic N at around 398.5, 399.0, 400.4, and 401.9 eV, respectively.35 Figure 2 (B and D) shows the Co2p spectra of ZIF-67 and ZIF-67-700, respectively. As reported previously, the XPS value for the 2p3/2 peak in cobalt oxide was above 780 eV, while the binding energy of Co0 was in the range of 778 - 778.5 eV.32 Co2p3/2 spectrum of ZIF-67-700 shows the peaks at around 782.5, 780.4 and 778.9 eV (Figure 2 D), suggesting the presence of Co2+, Co3+ and Co0 and further Co, CoO, Co3O4 or Co-Nx sites in the sample, which was consistent with the observation form XRD results.

Figure 2. High resolution N1s (A) and Co2p (B) XPS spectra of ZIF-67 sample, and N1s (C) and Co2p (D) XPS spectra of ZIF-67-700 sample.

Electrochemical Properties of Co/N/C Nanomaterials. The electrocatalytic activities of the as-prepared Co/N/C nanomaterials with ZIF-67 as the precursor were investigated with RRDE voltammetry in aCSF. As depicted in Figure 3, bare GC electrode shows a very negative onset potential (about -0.6 V) for ORR, suggesting its low electrocatalytic activity toward ORR. In contrast, the Co/N/C catalysts all show positively shifted reduction potentials for ORR, of which the Co/N/C catalyst prepared at 700°C (i.e., ZIF-67-700) exhibits 10


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the most positive onset and half-wave potentials (red curve, lower panel, Figure 3 A). Among samples tested, ZIF-67-700 shows the lowest ring current during the ORR process (red curve, upper panel, Figure 3 A), indicating the best electrocatalytic performance of this catalyst. Electron transfer number of ORR at bare and various catalysts modified GC electrodes was displayed in Figure 3 B. The number of transferred electrons (n) was calculated with the RRDE curves with the equation, n = 4ID/(ID + IR/N). Where, ID and IR stand for disk and ring currents, respectively. N stands for collection efficiency and was calculated as 0.33. These results reveal that Co/N/C catalysts facilitate ORR mostly into water without less formation of toxic intermediate H2O2. In the light of reduction potentials and current for ORR, ZIF-67-700 exhibits the best electrocatalytic activity among the samples tested. Therefore, it was selected as the electrocatalyst for subsequent experiments.

Figure 3 (A) RRDE voltammograms recorded with bare GC (black curve) and electrodes modified with Co/N/C catalyst prepared at different temperatures (color curves). Scan rate at disk electrodes, 10 mV s-1 (lower panel). The ring current for the oxidation of H2O2 was recorded constantly on Pt ring electrode at +0.5 V (upper panel). (B) Electron transfer number of ORR obtained at bare (black curve) and Co/N/C-modified (color curves) GC electrodes at different potentials. The RRDE experiments were conducted in the O2-saturated aCSF (pH 7.4). Rotation rate of the electrode, 500 rpm.

Electrophoretic Deposition of ZIF-67-700 onto CFEs. Electrophoretic deposition is a useful technique to form uniform thin films onto conducting solid substrate.36-37 In the deposition process, electrically charged particles tend to transport and deposit onto the surface of an electrically conductive substrate with the driving 11



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force of an electric field. In order to achieve uniform coating of ZIF-67-700 catalyst onto CFE, electrophoretic deposition was employed here. When a potential of -1.0 V was applied between CFE and Pt electrode, ZIF-67700 catalyst with positive charge was effectively deposited on the surface of CFEs (Figure 4 A). The thickness of ZIF-67-700 catalyst was also easily controlled by changing the electrophoresis time; with increasing the deposition time, the surface coverage of ZIF-67-700 catalyst increased (Figure 4 B-E). With the deposition of 100 s, the surface coverage of the ZIF-67-700 catalyst was still low and parts of the CFEs were exposed. When the deposition time was prolonged as 300 s, the surface was totally covered with ZIF-67-700 catalyst (Figure 4 E). As revealed in Figure 4 G, the bare CFE is smooth with diameter of about 7 μm. After a 300-s deposition, the total diameter of the electrode increased to about 10 μm (Figure 4 H). Such a thickness of ZIF-67-700 catalyst film (~1.5 μm) on CFE was found to be stable for in vivo detection of O2, as demonstrated below.

Figure 4. (A) Schematic illustration of ZIF-67-700-CFEs fabrication by electrophoretic deposition. The microscope pictures of bare CFE (B) and ZIF-67-700-CFEs with different electrophoretic deposition times of (C) 100 s, (D) 200 s, (E) 300 s, and (F) 400 s. SEM images of bare carbon fiber (G) and ZIF-67-700-CF (H). Scale bar: 20 μm (B-F) and 5 μm (G-H).

Selectivity and Linearity for Oxygen Detection. As we know, there are many electrochemically active chemical species in the cerebral system. During electrochemical detection, some neurochemicals like AA, DA, 5-HT, DOPAC and UA normally interfere with the detection. As shown in Figure 5 A, when the ZIF-67-700CFEs were polarized at a potential of -0.5 V, the addition of the neurochemicals mentioned above did not generate discernible current response, indicating these species had no interference with the detection of O2. The 12


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high selectivity of the ZIF-67-700-CFE toward O2 well validities its application for selective O2 monitoring in the cerebral system. Besides this, we also investigated the response of the ZIF-67-700-CFEs towards O2. As shown in Figure 5 B, the current increases with increasing O2 concentration and is proportional with O2 concentration with a linear coefficient of 0.9994 and a sensitivity of 0.0012 μA·μM-1.

Figure 5. (A) Typical amperometric response recorded at the ZIF-67-700-CFE toward AA (400 μM), DA (10 μM), DOPAC (20 μM), UA (50 μM), 5-HT (10 μM), and O2 (100 μM). The electrode was polarized at -0.5 V vs. Ag/AgCl (aCSF, pH 7.4). (B) Typical cyclic voltamograms obtained at the ZIF-67-700-CFE in aCSF (pH 7.4) saturated with O2 (red curve), ambient air (blue curve), and N2 (black curve). Scan rate, 50 mV s-1. Inset, the linear relationship between current response and the concentration of O2.

In Vivo Monitoring of Hippocampus O2. Before in vivo measurements, the ZIF-67-700-CFEs were further coated with Nafion to stabilize the catalyst onto the electrode surface and minimize the non-specific fouling mainly from protein.25 As typically displayed in Figure 6 A, after implantation for a few seconds, the reduction current of O2 remained almost unchanged during 1 h of continuous measurement, suggesting that the ZIF-67-700-CFE was quite stable for continuous monitoring of O2 in rat hippocampus in vivo. The basal level of O2 in rat hippocampus was measured as 30 ± 10 μM (n = 3), which was in good agreement with the values reported previously.25 To further validate the ZIF-67-700-CFEs for in vivo O2 monitoring, we used the prepared electrodes to real-time record the O2 fluctuation when the animals respire O2, N2, and CO2. As shown in Figure 6 B and C, breathing pure O2 leads to the fast increase of the reduction current by ca. 167% at the ZIF-67-700-CFE, suggesting the increase in the O2 level in rat hippocampus, while breathing pure N2 or CO2 noticeably decreases 13



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the current response by ca. 67% and 60%, respectively, indicating the decrease in the O2 level in rat hippocampus. These observations demonstrate that the ZIF-67-700-CFEs have a quick response time, which eventually enabled a real-time monitoring of the fluctuation of O2 in the brain caused by the fast transport of inhaled gases.

Figure 6. In vivo continuous amperometric response recorded with the ZIF-67-700-CFE to O2 in rat hippocampus (A) and amperometric responses recorded in rat hippocampus when the rats were exposed to pure O2 (B), N2 (B), or pure CO2 (C). The microelectrode was implanted into the rat hippocampus and polarized at 0.5 V vs. Ag/AgCl (aCSF, pH 7.4).

CONCLUSIONS In conclusion, we have for the first time demonstrated that Co/N/C nanomaterials can be used for chemical sensing applications. The Co/N/C catalyst is electrophoretically deposited onto carbon fiber electrodes to form an electrochemical sensor for real-time sensing of brain O2. The catalyst shows a high electrocatalytic efficiency toward ORR through a four-electron process without the generation of toxic intermediate H2O2. As a noble metal-free electrochemical sensor, the ZIF-67-700-CFEs possesses high selectivity, stability and sensitivity for in vivo monitoring of O2. This study not only provides a new opportunity for the application of functional M/N/C nanocomposites, but also builds a novel analytical protocol for in vivo analysis of chemical species in the central nervous system. ACKNOWLEDGEMENTS Financial support from the National Natural Science Foundation of China (21705155 for W. M., 21790390, 21790391, 21435007, 21621062 for L. M., and 21671135 for H. W.), Shanghai Municipal Education Commission (14ZZ128 for H. W) and the National Basic Research Program of China (2016YFA0200104), and the Chinese Academy of Sciences (XDB30000000,QYZDJ-SSW-SLH030) are acknowledged. 14


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Graphical Abstract

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Electrophoretically Sheathed Carbon Fiber Microelectrodes with Metal

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