Controllable and Reproducible Sheath of Carbon Fibers with Single

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Controllable and Reproducible Sheath of Carbon Fibers with Single-Walled Carbon Nanotubes through Electrophoretic Deposition for In Vivo Electrochemical Measurements Tongfang Xiao, Yanan Jiang, Wenliang Ji, and Lanqun Mao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00303 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

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Analytical Chemistry

Controllable and Reproducible Sheath of Carbon Fibers with Single-Walled Carbon Nanotubes through Electrophoretic Deposition for In Vivo Electrochemical Measurements Tongfang Xiao,1,2 Yanan Jiang,1,2 Wenliang Ji,1 Lanqun Mao1,2,* 1

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

Biosystems, Institute of Chemistry, the Chinese Academy of Sciences, Beijing 100190, China. 2

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

Corresponding Author. E-mail: [email protected], Fax: (86)-10-62559373.

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ACS Paragon Plus Environment

Analytical Chemistry 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

ABSTRACT The unique electronic and chemical structures of carbon nanotubes (CNTs) have well enabled their applications in electrochemistry and electroanalytical chemistry, however, the difficulty in reproducibly confining CNTs onto substrate electrode, particularly onto microelectrodes, still remains to be addressed. In this study, we develop a method to reproducibly confine single-walled carbon nanotubes (SWNTs) onto carbon fiber microelectrodes (CFEs) with electrophoretic deposition (EPD) for in vivo measurement of ascorbate. Under 2.5 V, acid-treated SWNTs are uniformly deposited on CFEs. After thermal treatment at 300°C followed by electrochemical treatment in 0.5 M H2SO4, the SWNT-sheathed CFEs exhibit a good activity to accelerate the electrochemical oxidation of ascorbic acid (i.e., ascorbate, in a neutral solution) at an onset potential of -0.15 V vs. Ag/AgCl and could in vivo selectively detect ascorbate. The controllable procedures employed for EPD and pretreatment avoid the deviation in the conventional manual modification methods such as drop-casting and hand-rolling previously used for confining SWNTs onto electrode surface. With the electrodes prepared here, we find that level of extracellular ascorbate in rat cortex increases by 20.4 ± 4.8% (n = 4), relative to its basal level, within 9 min after infusion of kainic acid into hippocampus to evoke epilepsy. This study offers a reproducible method to prepare SWNT-sheathed CFEs for in vivo monitoring ascorbate that would largely facilitate future studies on neurochemical processes of ascorbate in various physiological and pathological events.

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As one of the most important neurochemicals, ascorbate plays various roles in central nervous system (CNS).1-3 It has been generally accepted that ascorbate is one of neuroprotective antioxidants and free radical scavengers in oxidative stress processes such as energy failure, anoxic depolarization, and ischemic stroke.4-8 There are also evidences suggesting the non-antioxidant function of ascorbate as an enzyme co-factor participating in biosynthesis of catecholamines, carnitine, amino acids and certain peptide hormones and as a neuromodulator of both dopamine- and glutamate-mediated neurotransmission.9-12 Very recently, we find that ascorbate could be released by vesicular exocytosis from adrenal chromaffin cell through single-cell amperometry.13 However, a reliable method for in vivo monitoring of ascorbate in the CNS remains very essential to understanding its roles in various physiological and pathological processes. As reported previously, ascorbate is readily oxidized electrochemically into dehydroascorbic acid (DHA) through a two-electron and one-proton process followed by irreversible hydrolysis of DHA to finally produce 2, 3-diketogulanic acid.14 By fully using the electrochemical property of ascorbate, others and we have developed electrochemical methods for in vivo monitoring of ascorbate.15,16 Compared with other methods for in vivo monitoring of neurochemicals such as high performance liquid chromatography (HPLC) coupled with offline electrochemical detection, in vivo electrochemistry with tissue-implantable carbon fiber electrodes (CFEs) remains remarkable in terms of its high spatiotemporal resolution and good capability to accurately measuring chemically instable species such as ascorbate.17-21 However, the sluggish electron transfer property of ascorbate at pristine CFEs, presumably caused by electrode fouling with the final oxidation product of ascorbate (i.e., 2, 3-diketogulanic acid), unfortunately renders a large overpotential to the oxidation process and subsequently poor selectivity to the electrochemical detection of ascorbate.22 At most kinds of frequently used electrodes, ascorbate is an inner-sphere redox species with electron-transfer kinetics sensitive to surface chemistry of electrode.23,24 Thus, tuning surface chemistry and thereby interaction between ascorbate and electrode would pave an effective avenue to acceleration of electron-transfer kinetics of the oxidation of ascorbate.25 To this end, preactivating carbon electrodes, particularly CFEs, with different techniques such as electrochemical methods led to a large increase in the electrode activity for the oxidation of ascorbate and thus enabled the as-prepared electrodes to selectively monitor ascorbate in vivo.15,26 However, it remains practically difficult to reproducibly produce the same surface chemistry at the as-preactivated electrodes even under the same preactivation conditions, resulting in 3



variation in the electrode activity of the as-preactivated electrodes toward ascorbate because of its surface chemistry-dependent electron-transfer property.27 To overcome this limitation, externally sheathing CFEs with an electrocatalyst would pave an more effective route to acceleration of electron-transfer kinetics of ascorbate as compared with engineering the surface chemistry inherent in CFEs through various preactivation methods.28-33 This is because the former method is more experimentally controllable and reproducible compared with the latter methods. In our early studies, we have found that externally sheathing CFEs with CNTs largely facilitates the oxidation of ascorbate, enabling selective measurements of ascorbate in CNS in vivo.16,34 In this case, the deviation in CFEs from different sources is well eliminated because electrochemical reaction occurs at CNTs, and CFEs are simply used as substrate electrode, however, how to reproducibly sheath CFEs with CNTs to form CNT-sheathed CFEs remains to be an important issue for in vivo electrochemical monitoring of ascorbate. In our previous study, we used a drop-casting method to prepare CNT-modified CFEs by manually rolling CFEs in one drop of CNT dispersion.16 While the as-prepared electrodes accelerate electron-transfer kinetics for ascorbate oxidation as compared with pristine CFEs and could thus selectively monitor ascorbate in vivo, manual modification of CNTs onto CFEs normally led to electrode-to-electrode deviation and un-uniform surface coverage of CNTs onto CFEs. As one step forward, we used vertically aligned carbon nanotube-sheathed CFEs (VACNT-CFEs) as microelectrodes for in vivo monitoring of ascorbate.34 While the preparation of VACNT-CFEs well avoided the manual modification procedures, it required complicated conditions to grow CNTs onto carbon fibers, offering limitation in mass preparation of tissue-implantable CFEs for in vivo measurement of ascorbate. As a continuous attempt in this area, we present herein a controllable and reproducible method to sheath CFEs with CNTs with electrophoretic deposition (EPD) for in vivo monitoring of ascorbate. Under electric field, CNTs are forced to move towards and finally deposit on the surface of CFEs to form CNT-sheathed CFEs. After thermal and electrochemical pretreatments, the CNT-sheathed CFEs show improved electron-transfer kinetics toward ascorbate oxidation and are capable to selectively monitor ascorbate in vivo in the CNS. Moreover, the EPD method demonstrated here is easily adoptable by non-electrochemists, which would advance the research on the role of ascorbate in some physiological and pathological processes.

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EXPERIMENTAL SECTION Reagents and Solutions. Ascorbate, dopamine (DA), uric acid (UA), 3, 4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT), epinephrine (E) and norepinephrine (NE) were all purchased from Sigma and used as supplied and the solutions were prepared just before use. Artificial cerebrospinal fluid (aCSF) was prepared by mixing NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM), and CaCl2 (1.1 mM) into Milli-Q water and the solution pH was adjusted to pH 7.4. Three types of SWNTs were purchased from different sources, i.e., SWNT-1 (1 - 2 nm in diameter, 5 - 15 µm in length, from Shenzhen Nanotech Port Co., Ltd. (Shenzhen, China)), SWNT-2 (1 - 2 nm in diameter, 0.5 - 2 µm in length, from Dk Nano technology (Beijing, China)), and SWNT-3 (0.7 - 2.5 nm in diameter, 0.5 - 10 µm in length, from BuckyUSA Inc. (Houston, USA)). Double-walled carbon nanotubes (DWNTs) (2 - 4 nm in diameter, 5 - 15 µm in length) and multi-walled carbon nanotubes (MWNTs) (10 - 20 nm in diameter, 10 - 30 µm in length) were both purchased from Dk Nano Technology (Beijing, China). Kainic acid was purchased from Sigma and dissolved in aCSF before local injection into rat hippocampus. Other chemicals were of at least analytical reagent and used without further purification. All aqueous solutions were prepared with Milli-Q water. Unless stated otherwise, all experiments were carried out at room temperature. Preparation of CFEs and CNT-Sheathed CFEs. CFEs were fabricated as described previously.35 To investigate the electroactivity variation of CFEs prepared with CFs from different sources, three type of CFs were used to prepare CFEs; Type-1 (7 µm in diameter, Tokai Carbon Co., Tokai, Japan); Type-2 (5 µm in diameter, ProCFE, Dagan, Minneapolis, MN, USA), and Type-3 (7 µm in diameter, Goodfellow, Cambridgeshire, England). A single CF was attached to copper wire and the wire was inserted into glass capillary (o.d. 1.5 mm, length 100 mm) that was pulled on a microelectrode puller (WD-1, Chengdu Instrument Factory, Sichuan, China) with a sharp fine tip at 30-50 µm in diameter. Both ends of the capillary were then sealed with epoxy resin with 1:1 ethylendiamine and the capillary with CF-attached Cu wire was dried at 100 °C for 2 h. Before use, the exposed CF was cut to 200-500 µm by a surgery scalpel under a microscopy. To prepare SWNT-sheathed CFEs, 0.1 g SWNTs was suspended into 100 mL concentrated HNO3 and H2SO4 (1:3 volume ratio) mixture. The suspension was ultrasonicated in water bath for 3 h at 40 ºC, washed 5



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with Milli-Q water to neutral, and finally dried in vacuum drying oven overnight. To deposit SWNTs onto CFEs to form the SWNT-sheathed CFEs with EPD, SWNTs were dispersed into Milli-Q water and the dispersion was sonicated to give a homogeneous dispersion (2 mg/mL). EPD was performed in the dispersion with CFEs as anode and Pt wire as cathode with a distance of 1.5 mm between two electrodes. A voltage of 2.5 V was applied between CFEs and Pt wire with different time duration to control the thickness of SWNT film on CFEs. The as-prepared SWNT-sheathed CFEs were thermally treated in electric furnace under nitrogen atmosphere at 300 °C for 2 h followed by electrochemical treatment in 0.5 M H2SO4, first with a constant potential at +2.0 V for 30 s, then at -1.0 V for 10 s, and finally with cyclic voltammetry in 0.5 M H2SO4 within a potential range from 0 to 1.0 V at a scan rate of 0.1 V s-1 until a stable cyclic voltammogram was obtained. Apparatus

and

Measurements.

Electrochemical

measurements

were

performed

on

a

computer-controlled electrochemical analyzer (CHI 730D, Shanghai, China). The CFEs and SWNT-sheathed CFEs were used as working electrode and a Pt wire as counter electrode. For both in vitro and in vivo electrochemical measurements, a tissue-implantable Ag/AgCl microelectrode was used as reference electrode. The reference electrode was prepared by polarizing Ag wire at +0.6 V in 0.1 M hydrochloride acid for ca. 30 min and then inserting the wire into a glass capillary. Agar (30 mg/mL) thermally melted in 4.0 M KCl solution was first sucked from the fine end of the capillary (o.d., ca. 30 µm) and then cooled under room temperature to work as salt bridge to separate inner aCSF that was infused from the larger open end of the capillary. The larger open end of the capillary was finally sealed with epoxy with the exposed Ag wire as conductor. To demonstrate the dependence of electrode reactivity on the conditions employed for electrode pretreatment, CFEs were subject to electrochemically preactivated under different conditions; 1) in 1.0 M KOH at a constant potential of +1.5 V for 80 s and then with cyclic voltammetry within a potential range from 0.0 to +1.0 V at a scan rate of 0.1 V s-1 in 1.0 M KOH until a stable cyclic voltammogram was obtained; 2) in 0.5 M H2SO4 at a constant potential of +2.0 V for 30 s, at -1.0 V for 10 s, and finally with cyclic voltammetry in 0.5 M H2SO4 within a potential range from 0.0 to +1.0 V at a scan rate of 0.1 V s-1 until a stable cyclic voltammogram was obtained. Scanning electron microscopy (SEM) was performed on a Hitachi S4300-F microscope (Japan). In Vivo Experiments. Adult male Sprague-Dawley rats (300 - 350 g) were purchased from Beijing Vital 6


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River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animals were housed on a 12 h:12 h light-dark schedule with food and water ad libitum. The use and care of animals were approved and directed by the Institutional Animal Care and Use Committee of National Center for Nanoscience and Technology of China. The animals were anesthetized with isoflurane (4% induction, 2% maintenance) through a gas pump (RWD R520, Shenzhen, China) and positioned onto a stereotaxic frame during in vivo experiment. The SWNT-sheathed CFE was implanted into rat right cortex (AP = -2.0 mm, L = -2.0 mm from bregma, V = 1.7 mm from dura) using standard stereotaxic procedures. The Ag/AgCl reference electrode was positioned into the dura of brain. Platinum wire was embedded in subcutaneous tissue on the brain and used as counter electrode. Local drug delivery by exogenous microinfusion of kainic acid was performed with silica capillary tubes (4 cm length, 50 µm i.d., 375 µm o.d.). Infusion solution was delivered from a gastight syringe and pumped through tetrafluoroethylene hexafluoropropene (FEP) tubing by a microinjection pump (CMA 100, CMA Microdialysis AB, Stockholm, Sweden). Local microinfusion was applied to rat right hippocampus (AP = -5.0 mm, L = -5.0 mm from bregma, V = 5.5 mm from dura) at a perfusion rate of 0.5 µL min-1 for 5 min. The SWNT-sheathed CFE was polarized at a constant potential of +0.05 V for in vivo amperometric measurement of ascorbate in rat cortex.

RESULTS AND DISCUSSION Electrochemical Oxidation of Ascorbate at Preactivated CFEs. CFEs have been previously used for in vivo monitoring of ascorbate in rat brain.15,36,37 To achieve the selectivity, CFEs were electrochemically preactivated in acidic or basic media, whereas the activity for the electrochemical oxidation of ascorbate varies with the conditions employed for the electrode preactivation and sources of carbon fibers.38-40 To demonstrate this variation, we chose three types of carbon fibers to prepare CFEs (i.e., Type-1, Type-2, and Type-3 CFEs) and preactivated the CFEs electrochemically in acidic (i.e., 0.5 M H2SO4) or basic (1.0 M NaOH) solution. Figure 1 compares cyclic voltammograms (CVs) obtained at pristine (no preactivation) and preactivated CFEs in different media for the oxidation of ascorbate in aCSF (pH 7.4). Without electrochemical preactivation in acidic or basic solutions, the pristine CFEs displayed a tailed current response for ascorbate oxidation with variation in the onset potentials for the oxidation of ascorbate (A, B, C, upper panel). Electrochemical preactivation in 0.5 M H2SO4 accelerates the electron-transfer rate of ascorbate oxidation at 7



Type-1 CFE

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Figure 1. Typical CVs obtained at CFEs in aCSF (pH 7.4) in the absence (dashed curves) and presence (solid curves) of 400 µM ascorbate. The CFEs were prepared with CFEs from different sources (i.e., Type-1 (A), Type-2 (B), and Type-3 (C)) and electrochemically pretreated in 1.0 M NaOH or 0.5 M H2SO4. Scan rate was 0.05 V s-1.

the Type-1 and Type-3 CFEs (A, C, middle panel), which was observed with the negative shift of the onset potential for ascorbate oxidation, but does not obviously alter the electrochemical oxidation at the Type-2 CFE (B, middle panel). The similar trend was also observed for the electrochemical preactivation of Type-1 and Type-3 CFEs in 1.0 M NaOH solution (A, C, lower panel), suggesting that electron-transfer kinetics of ascorbate oxidation on Type-1 and Type-3 CFEs was remarkably improved by electrochemically preactivating CFEs in 1.0 M NaOH. Interestingly, for Type-2 CFE (B), the electrochemical preactivation either in acidic or basic solution did not greatly alter the electron-transfer kinetics for the oxidation of ascorbate. This is also different from the cases of Type-1 and Type-3 CFEs, in which the electron-transfer kinetics of ascorbate oxidation largely depends on the conditions employed for the preactivation (A, C). These results show that the electrode activity for the oxidation of ascorbate differs remarkably at the CFEs prepared with carbon fibers from different sources even the CFEs were preactivated under the same conditions. It is known that ascorbate is one of inner-sphere species, of which electron-transfer kinetics is highly sensitive to the density of electronic states (DOS), surface structure, and surface chemistry of the electrode.24,25 Electrochemical preactivation in acidic or basic media could change the surface chemistry such as oxygen functional group ratio, surface structure (e.g., defects), and DOS as well.39,40 However, these changes are associated with the

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source of carbon fibers and the conditions employed for the preactivation, as demonstrated here. Thus, electrochemical preactivation applied to CFEs normally requires a trial-and-error procedure to acquire optimal electrochemical response, depending on the analyte of interest and source of carbon fibers, resulting in electrode-to-electrode deviation in the electrochemical response and laboratory-to-laboratory variation in the conditions for electrode preactivation. Electrochemical Oxidation of Ascorbate at SWNT-Sheathed CFEs with EPD. Figure 2 displays SEM images of the SWNT-sheathed CFEs prepared by EPD at a potential of 2.5 V for different time. The surface coverage of SWNTs on CFEs increases with increasing the time employed for EPD. As the EPD time prolongs to 30 s, the thickness of SWNT coating reaches ca. 4 µm and CFEs were totally and uniformly sheathed with SWNTs. Other two types of SWNTs from different sources were also successfully deposited onto CFEs by EPD after the pretreatment in the acidic solution (data not shown). Note that, we have tried to sheath CFEs with MWNTs and DWNTs with the same EPD method and found that these nanotubes were not deposited onto CFEs even we increased the potential to 3.0 V, possibly due to the slow electromigration rate of these nanotubes under the conditions employed here.41 Although we have previously prepared CNT-modified CFEs or glassy carbon electrodes for in vivo and online electrochemical measurement of ascorbate with a drop-casting method and by vertical alignment of CNTs onto electrodes,3,16 the EPD method A

B

3 µm

3 µm

C

D 500 nm

3 µm

5 µm

Figure 2. SEM images of pristine (A) and SWNT-sheathed CFEs by EPD at a potential of 2.5 V for 5 s (B), 15 s (C), and 30 s (D). Inset in D, enlarged SEM image of SWNT-sheathed CFE.

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developed here remains to be a great advance in preparing SWNT-sheathed CFEs because of its capability to uniformly form SWNT film onto CFEs with a controlled film thickness. We next investigated the electrochemical property of the SWNT-sheathed CFEs toward the oxidation of ascorbate in aCSF. Before electrochemical studies, the SWNT-sheathed CFEs were thermally treated at 300 °C under nitrogen atmosphere for 2 h followed with electrochemical treatment with a constant potential of +2.0 V for 30 s and -1.0 V for 10 s in 0.5 M H2SO4 to enable the oxidation of ascorbate at a low potential. Figure 3 shows CVs obtained at the SWNT-sheathed CFEs for the oxidation of ascorbate in aCSF. As shown in Figure 3 A, B and C, the CFEs sheathed with SWNTs from different sources show almost the same onset potentials (ca. -0.15 V) for the oxidation of ascorbate. Moreover, the potentials for the current reaching near steady-state current (ca. -0.05 V) were also almost the same at three electrodes. These results demonstrate that the CFEs sheathed with SWNTs well accelerate the electron-transfer rate of ascorbate, which was consistent with our previous observation,16 and that the CFEs sheathed with SWNTs show almost the same electrochemical activity toward the oxidation of ascorbate at a low potential, regardless of the source of SWNTs. This is very different from CFEs, of which the electrochemical property varies largely with the source of carbon fibers as shown in Figure 1. Note that, we did not observe the occurrence of ascorbate oxidation at such a low onset potential at CFEs when we thermally and electrochemically pretreated pristine CFEs under the same conditions (data not shown), showing that the oxidation of ascorbate occurs at the SWNTs at the SWNT-sheathed CFEs. In addition, we found that the electrode-to-electrode variation was also minimal; we have prepared ten SWNT-sheathed CFEs with SWNTs from the same source and investigated their voltammetric responses toward ascorbate (Figure 3D). At all electrodes, the oxidation of ascorbate commences at (-150 ± 6) mV, and reaches steady-state current at (-70 ± 7) mV with a half-peak potential of ca. -110 mV versus Ag/AgCl (aCSF), which was close to the formal redox potential of ascorbate (ca. -190 mV versus Ag/AgCl (aCSF)).42 These ten electrodes also showed nearly reversible potential responses (|E3/4 - E1/4| = (40 ± 2) mV) and the same current response (ca. 120 nA) for the oxidation of 0.4 mM ascorbate, demonstrating that the use of EPD method to prepare the SWNT-sheathed CFEs well avoid the person-to-person and electrode-to-electrode deviations associated with the CNT electrodes prepared by manual modification of CNTs onto substrate electrode.

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Figure 3. Typical CVs obtained at CFEs sheathed with SWNTs from different sources (A, B, and C), and at ten CFEs sheathed with SWNTs from the same source (D). Prior to the electrochemical studies, the SWNT-sheathed CFEs were thermally and electrochemically pretreated. Electrochemical studies were performed in aCSF (pH 7.4) in the absence (dashed curves) and presence (solid curves) of 400 µM ascorbate. Scan rate was 0.05 V s-1.

Moreover, the SWNT-sheathed CFEs exhibited improved stability (A) and high selectivity (B) toward ascorbate without the interference from other electroactive species in the CNS. As shown in Figure 4A, the SWNT-sheathed CFE was quite stable for the measurement of ascorbate, with oxidation current decreases by only 4.5% after continuous measurement of 0.5 mM ascorbate for 30 min (red curve). Compared with the SWNT-sheathed CFE, pristine and electrochemically preactivated CFEs were not stable for the measurement of ascorbate and the currents for ascorbate oxidation decrease by more than 20% after continuous measurement of 0.5 mM ascorbate for 30 min (black, olive, and blue curves). In addition, the excellent electrochemical properties of SWNT-CFEs make it possible to selectively monitor ascorbate without interference from other electroactive species in the CNS, as shown in Figure 4B. These properties, along with 11



the reproducible and controlled feature of EPD method employed for the electrode preparation, substantially enable the SWNT-sheathed CFEs particularly useful for in vivo monitoring of ascorbate in CNS.

Figure 4. (A) Typical amperometric response recorded with different CFEs for 500 µM ascorbate with the SWNT-sheathed CFE (red curve), pristine CFE (i.e., without preactivation) (black curve), and CFEs electrochemically preactivated in 1.0 M KOH at +0.05 V (olive curve) or acidic media at +0.30 V (blue curve). I0 and I were current values recorded at starting time and given time, respectively. (B) Typical amperometric response recorded with the SWNT-sheathed CFE at +0.05 V vs. Ag/AgCl (aCSF) toward 50 µM DA, 20 µM NE, 50 µM 5-HT, 20 µM E, 50 µM UA, 50 µM DOPAC and 200 µM ascorbate.

Towards In Vivo Monitoring of Ascorbate. Prior to in vivo monitoring of ascorbate in the CNS, we investigated the in vivo stability of the as-prepared SWNT-sheathed CFEs by implanting the electrode into rat cortex to continuously monitor ascorbate. As typically shown in Figure 5A, the current response stabilized a few minutes after the electrode was implanted into rat cortex and did not change evidently during 1 h of in vivo experiment in rat cortex, demonstrating the good stability of the electrode for in vivo measurements of ascorbate. With these properties, the SWNT-sheathed CFE was employed to investigate ascorbate fluctuation in rat cortex during epilepsy evoked by kainic acid. Kainic acid is a natural marine acid and a potent neuroexcitatory amino acid agonist for kainate receptors that acts by activating receptors for glutamate and evokes seizures.43 As typically shown in Figure 5B, the local injection of kainic acid into rat hippocampus led to an increase in the concentration of extracellular ascorbate in rat cortex by 20.4 ± 4.8% (n = 4), in relative to the basal level (181 ± 21) µM (n = 4), which was almost consistent with the previous reports.34,44 While more evidence is needed to understand the mechanism underlying the increase of extracellular ascorbate, this observation suggests that the increase in the extracellular ascorbate in the rat cortex is one of the chemical 12


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consequence of kainic acid-induced epilepsy. 12

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Figure 5. (A) Typical amperometric response recorded in vivo with the SWNT-sheathed CFEs for in vivo monitoring of extracellular ascorbate in rat cortex. (B) Typical amperometric current response recorded in vivo with the SWNT-sheathed CFE before and after local microinfusion of 2.5 µL of kainic acid into the rat hippocampus. The electrode was polarized at + 0.05 V vs. Ag/AgCl (aCSF). Note that, the electrodes used in A and B had different length and thus the current responses were not comparable.

CONCLUSIONS In summary, we have for the first time developed an effective EPD method to reproducibly and controllably prepare SWNT-sheathed CFEs for in vivo electrochemical measurement of ascorbate. After thermal and electrochemical pretreatment, the SWNT-sheathed CFEs exhibit excellent activity toward oxidation of ascorbate with the activity independent of the source of CFEs and SWNTs, high selectivity and good stability for the measurement of ascorbate. These properties eventually facilitate in vivo electrochemical monitoring of fluctuation of ascorbate in rat brain, which would advance future researches on understanding the roles of ascorbate in some physiological and pathological processes.

AUTHOR INFORMATION Corresponding Author *

Email: [email protected]. Fax: (+86)-10-62559373.

Notes The authors declare no competing financial interest. 13



ACKNOWLEDGEMENTS We acknowledge financial support from the National Natural Science Foundation of China (21790390, 21790391, 21621062 and 21435007 for L. Mao), the National Basic Research Program of China (2016YFA0200104), and the Chinese Academy of Sciences.

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For TOC only

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Controllable and Reproducible Sheath of Carbon Fibers with Single

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