Protein Pretreatment of Microelectrodes Enables in Vivo

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Protein Pre-treatment of Microelectrodes Enables In Vivo Electrochemical Measurements with Easy Pre-calibration and Interference-Free from Proteins Xiaomeng Liu, Meining Zhang, Tongfang Xiao, Jie Hao, Ruixin Li, and Lanqun Mao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01476 • Publication Date (Web): 21 Jun 2016 Downloaded from http://pubs.acs.org on June 22, 2016

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Analytical Chemistry

Protein Pre-treatment of Microelectrodes Enables In Vivo Electrochemical Measurements with Easy Pre-calibration and Interference-Free from Proteins Xiaomeng Liu,† Meining Zhang,†,* Tongfang Xiao,‡ Jie Hao,‡ Ruixin Li,† Lanqun Mao‡,* †

Department of Chemistry, Renmin University of China, Beijing 100872, China.

‡

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical

Chemistry for Living Biosystems, Institute of Chemistry, the Chinese Academy of Sciences (CAS), Beijing 100190, China.

*

Corresponding Authors. Fax: +86-10-62559373;

E-mail: [email protected]; [email protected]. 1

ACS Paragon Plus Environment


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ABSTRACT: In vivo electrochemistry is one powerful strategy for probing brain chemistry. However, the decreases in sensitivity mainly caused by the adsorption of proteins onto electrode surface in short-term in vivo measurements unfortunately render great challenges in both electrode calibration and selectivity against the alternation of proteins. In this study, we observe that the pre-treatment of carbon fiber microelectrodes (CFEs) with bovine serum albumin (BSA) would offer a simple but effective strategy to the challenges mentioned above. We verify our strategy for dopamine (DA) with conventionally used CFEs and for ascorbate with our previously developed carbon nanotube-modified CFEs. We find that, in artificial cerebral spinal fluid (aCSF) solution containing BSA, the current responses of the microelectrodes equilibrate shortly and the results for pre-calibration carried out in this solution are found to be almost the same as those for the post-calibration in pure aCSF. This observation offers a new solution to electrode calibration for in vivo measurements with a technical simplicity. Furthermore, we find that the use of BSA pre-treated CFEs to replace bare CFEs would minimize the interference from the alternation of proteins in the brain. This study offers a new general and effective approach to in vivo electrochemistry with a high reliability and a simplified procedure.

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Introduction In vivo monitoring the dynamics of the changes neurochemicals becomes more and more important to unveil and understand brain activity and function.1-4 In the central nervous system, neurochemicals are released at neuro terminals, of which some of them can be real-time recorded with electrochemical methods.5-10 Due to its capability to track the dynamic change of neurochemicals with a high spatial and temporal resolution, in vivo electrochemistry has been of great concern both in chemistry and neuroscience communities.11-14 However, the adsorption of biomacromolecules, proteins in particular, on the surface of microelectrodes during short-term in vivo electrochemical recording inevitably results in the decrease in the sensitivity for in vivo measurements, which render great challenges to both electrode calibration and selectivity against the alternation of proteins in the proceed of implantation and brain activities.15-19 The general strategy employed for electrode calibration for in vivo measurements normally relies on calibrating electrodes after animal experiments, i.e., post-calibration.20-23 Besides its limitation in complexing the measurements with a risk in breaking the electrodes during the electrode withdrawing process from brain tissues, post-calibration is valid only on the condition that the process for the protein adsorption on electrode caused is quick and unchangeable in the time scale for in vivo measurements. That is, when the protein adsorption is relatively slow and changeable due to the alternation of proteins in brain environment over the time frame of in vivo experiments, post-calibration will be no longer suited. To avoid the problem inherent in post-calibration, some methods have been developed. These include in situ electrode calibration15 and hydrogenation of carbon microelectrode,24 of which coating the microelectrode surface with anti-fouling films, e.g., Nafion, base-hydrolyzed cellulose acetate (BCA), and polyenedioxythiophene (PEDOT)/Nafion, remains very popular.25-31 Nevertheless, Singh et al. systematically investigated the effects of three coatings materials, 3



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i.e., Nafion, BCA on CFE sensitivity, selectivity, and biofouling with monoamine neurotransmitter as the target analyte.25 They found that electrode responses to different coating processes and fouling were complex, with some coatings better suited for sensitivity and selectivity (i.e., Nafion), whereas others were better at preventing fouling (i.e., BCA or fibronectin). Furthermore, most of coating materials could, to some extent, recover sensitivity. Thus, the post-calibration is still needed to quantify in vivo data. In this sense, a new and general strategy is highly required for reliable pre-calibration for in vivo electrochemical measurements. In this study, we for the first time observe that the results obtained for pre-calibration of electrodes carried out in artificial cerebral spinal fluid (aCSF) containing bovine serum albumin (BAS) are almost the same as those for the post-calibration conducted in the general ways (i.e., in pure aCSF), demonstrating that electrode pre-calibration in aCSF containing BSA is valid and effective for in vivo measurements. In this case, the electrode/electrolyte interface formed in the medium employed here for electrode pre-calibration (i.e., in aCSF containing BSA) may be considered to be similar to that formed in real brain environment in short-term in vivo analysis, from the electrochemical point of view. Moreover, we also find that the BAS-pretreated CFEs bear a good selectivity against alternation of proteins. This study essentially demonstrates a new strategy to enable in vivo electrochemical measurements to be easily pre-calibrated and be interference-free from proteins.

EXPERIMENTAL SECTION Reagents

and

Solutions.

Dopamine

(DA),

ascorbate,

uric

acid

(UA),

3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT), bovine serum albumin (BSA) and cytochrome c (Cyt. c) were all purchased from Sigma and used as supplied, and the solutions were prepared just before use. Artificial cerebrospinal fluid (aCSF) 4


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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 was adjusted pH to 7.4. Multi-walled carbon nanotubes (MWNTs, 10-20 nm in diameter and < 2 µm in length) were purchased from Shenzhen Nanotech Port Co., Ltd, (Shenzhen, China). 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. Carbon Fiber Microelectrodes. Carbon fiber microelectrodes (CFEs) were fabricated as described previously.32 The exposed CF was cut to 200-500 µm for normal cyclic voltammetry and amperometric with a surgery scalpel under a microscopy. Prior to use in electrochemistry, the CFEs were first sonicated in acetone, 3 M HNO3, 1.0 M KOH, and distilled water sequentially, each for 3-5 min. Then, the electrodes were subject to electrochemical activation, first with potential-controlled amperometry at +2.0 V for 30 s and at -1.0 V for 10 s, and then with cyclic voltammetry in 0.5 M H2SO4 within a potential range from 0 V to 1.0 V at a scan rate of 0.1 V s-1 until a stable cyclic voltammogram was obtained. CFEs modified with MWNTs were prepared as reported previously.32 Briefly, MWNTs were dispersed into N, N-dimethylformamide and the mixture was sonicated to give a homogeneous dispersion (2 mg mL-1). One drop of the dispersion was applied onto a smooth glassy plate and CFEs were immersed and rolled into the droplet under a microscopy very carefully. After that, the electrodes (MWNT/CFEs) were taken out of the droplet and dried in ambient temperature. The BSA-treated electrodes (both CFEs and MWNT/CFEs) were prepared by first immersing the electrodes in aCSF containing 40 mg mL-1 BSA for 1 h, taken out of the solution, washed with water, and finally dried in ambient temperature. The Carbon fiber microelectrodes for fast-scan cyclic voltammetry (FSCV) were prepared as reported previously.33 Briefly, an 10 cm segment of carbon fiber (7 µm in diameter, Tokai 5



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Carbon Co., Tokai, Japan) was inhaled into an 10 cm long glass capillary (outer diameter: 1.5 mm; inner diameter: 0.89 mm; And then, the capillary was pulled to a CFE with a tapered tip in a horizontal puller

(P-2000, Sutter Instrument Co.). The exposed CF was cut to 100-300

µm with a surgery scalpel under a microscopy. Silicone rubber was back-filled into the tip of the CFE. In order to ensure that the silicone rubber completely filled the foremost tip, the CFE was fixed at a 2 mL centrifuge tube and centrifuged at 7000 rpm for 70 seconds. And the CFE was successfully prepared after the silicone rubber solidified at room temperature. The solidification time was about 24 h. The electrical junction was made by back-filling the capillary with 3 M KCl and inserting an Ag/AgCl wire. Apparatus and Measurements. Electrochemical measurements were performed with a computer-controlled Electrochemical Analyzer (CHI 760d, Shanghai, China). CFEs and MWNT/CFEs were used as working electrode, a platinum wire as counter electrode. In order to meet the requirement of in vivo measurements, we prepared a micro-sized Ag/AgCl electrode as reference electrode that could be implanted into brain tissue by first polarizing Ag wire (i.d. 1 mm) at +0.6 V in 0.1 M hydrochloride acid for ca. 30 min to make an Ag/AgCl wire and then inserting the Ag/AgCl wire into a pulled glass capillary, in which aCSF was sucked from the fine end of the capillary and used as the inner solution. The larger open end of the capillary was finally sealed with epoxy with the method mentioned above. To avoid the potential difference between in vivo and in vitro electrochemical measurements, the prepared micro-sized Ag/AgCl electrode was used in all electrochemical measurements. Two-electrode system applied for FSCV using Pinnacle Technology’s 8500 FSCV system. CFE for FSCV was used as working electrode, Ag/AgCl wire was used as reference electrode. Electric stimulation was generated using an isolator (ISO-Flex, AMPI, Israel). Surface wettability was determined using a contact angle measuring analyzer (JC2000D, Powereach, China) at ambient temperature. 6


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In Vivo Experiments. Adult male Sprague-Dawley rats (300-350 g) were purchased from Health Science Center, Peking University. The animals were housed on a 12:12 h light-dark schedule with food and water ad libitum. All procedures were approved by the Beijing Association on Laboratory Animal Care and the Association for Assessment and Accreditation of Laboratory Animal Care and performed according to their guidelines. Animal experiments were performed with a method described in our earlier work.34 Briefly, the animals were anaesthetized with chloral hydrate (345 mg/kg, i.p.) and positioned onto a stereotaxic frame. MWNT/CFEs and BSA-treated MWNT/CFEs were implanted into striatum (AP = 0 mm, L = 3 mm from bregma, V=4 mm from dura) using standard stereotaxic procedures.32 Stimulated DA release was recorded in the rat nucleus accumbens (NAc) in vivo by using an CFE and BSA-treated, as previously described.31 A bipolar stimulating electrode was implanted in medial forebrain bundle (MFB) (AP = -2.2 mm, L = 1.6 mm from bregma, V=7.3 mm from dura). The recording CFE electrode was implanted in NAc (AP = 2.0 mm, L = 1.5 mm from bregma, V = 6.5 mm from dura).32 A burst of pulse stimulations of 3 seconds at 60 Hz (± 350 µA, 2 ms per phase) was applied to trigger the DA release in NAc. The prepared micro-sized Ag/AgCl reference electrode was implanted from the dura of brain. Pt wire inserted into the brain muscle was used as counter electrode. Pre-calibration was performed before the microelectrode was implanted into brain tissue with successive adding DA and ascorbate into aCSF (pH 7.4) containing 40 mg mL-1 BSA. Post-calibration was performed in pure aCSF with successive addition of DA or ascorbaet after the microelectrodes was removed from the brain tissue.

RESULTS AND DISCUSSION Pre-calibration with BSA-pretreated Microelectrodes for Dopamine. As one of the most important neurotransmitters, DA plays a crucial role in various brain functions, such as 7



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reward-based behaviors, memory, addiction, and movement.35,36 DA is positively charged in neural conditions and electrochemically active and, as a result, could be real-time determined in vivo with electrochemical methods such as fast scan cyclic voltammetry and potential-controlled amperometric method.37-39 In order to eliminate the interference from the negatively charged ascorbate and electrode fouling resulted from the protein in brain, one negatively charged membrane, Nafion, was smartly introduced onto the surface of CFEs to achieve the sensitivity of DA and alleviate the fouling to some extent.26-29 We demonstrate our strategy with the BSA-treated CFEs by using DA as a target analyte. Figure 1 shows typical results for the calibration with CFEs for DA under different conditions. As displayed in Figure 1A, after the electrode has been implanted into striatum in rat brain for at least 2 h, the CFE still exhibits well-defined current responses to successive addition of DA in pure aCSF (red curve). The current response was linear with the concentration of DA (A, inset, red line), which forms a straightforward basis for post-calibration. The sensitivity of the electrode after in vivo implantation (i.e., 0.089 nA µM-1 ) was largely decreased as compared with that of the same electrode before in vivo implantation (i.e., 0.252 nA µM-1), as obtained as the slope of the pre-calibration curve (A, inset, black line). Such a decrease was ascribed by the electrode inactivation mainly caused by protein adsorption onto CFEs.25,40 Interestingly, we observed that, in aCSF containing BSA, the fresh CFEs (i.e., prior to brain-tissue implantation) exhibit well-defined current responses toward the successive addition of DA (Figure 1B, black curve). More importantly, such current responses were almost the same as those obtained with the same electrode in pure aCSF (i.e., containing no BSA) after the electrode was implanted into brain tissue for 2 hours, taken out for the brain, and washed with water (B, red curve). Accordingly, the calibration curve obtained under the former conditions (B, inset, black line), i.e., pre-calibration, was almost identical with that under the latter conditions (B, inset, red line), i.e., post-calibration. Moreover, as displayed in 8


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Figure 1C, the ratio of the sensitivity obtained with the post-calibration in pure aCSF (i.e., containing no BSA) (Spost) to that obtained with pre-calibration in aCSF containing BSA (Spre) was close to unity (i.e., 0.94 ± 0.07) (blue column). This value reasonably differs from that achieved with pure aCSF containing no BSA as the solution for pre-calibration (i.e., 0.31 ± 0.04) (red column). This observation strongly demonstrates that the use of aCSF containing BSA as the calibration solution to replace the pure aCSF normally used in the existing in vivo methods essentially offers a new and effective approach for electrode calibration for in vivo electrochemical measurements.

3 I / nA

I / nA

6

12

3

6

0 0

0

2

5

10

15

20

2

5

10

15

20

C / µM

400

600

0.6 0.4 0.2

0 200

C

0.8

1

0

1

C / µM

1.0

B

A

Spost/Spre

4 9

I / nA

18

I / nA

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


0

300

T/s

600 T/s

900

1200

0.0 0

1

2

3

Figure 1. A) Amperometric current responses obtained with CFEs toward successive addition of DA (each addition, 5 µM) in pure aCSF containing no BSA before (black curve) and after (red curve) in vivo implantation in striatum of rat brain for 2 h. Inset, pre- and post-calibration curves obtained with the CFEs before (black curve) and after (red curve) in vivo implantation. B) Amperometric current responses obtained with CFEs toward successive addition of DA (each addition, 5 µM) under different conditions: in pure aCSF containing 40 mg mL-1 BSA prior to in vivo implantation of CFEs (black curve), and in pure aCSF (i.e., containing no BSA) after in vivo implantation of the CFEs striatum of rat brain for 2 h (red curve). Inset, calibration curves obtained under both conditions. Potential +0.2 V (vs. Ag/AgCl). C) The ratio of sensitivity obtained in the pre-calibration of the CFEs in aCSF in the absence (red column) and presence (blue column) of 40 mg mL-1 BSA to that of post-calibration in pure aCSF after in vivo electrode implantation for 2 h.

The strategy demonstrated here for electrode calibration is relatively remarkable because, as mentioned above, although the post-calibration has been widely used for in vivo 9



measurements, this procedure essentially makes in vivo measurements technically complicated and requires a special caution to ensure that the electrodes are not broken when they are extracted from the brain tissues after in vivo measurements. Moreover, the post-calibration may not be suitable for the case when the sensitivity of the electrodes is altered during in vivo measurements. To understand the insights into the observation mentioned above, we first studied the effect of BSA on the current response of DA at CFEs. As typically shown in Figure 2, cyclic voltammetric (CV) result reveals that the presence of BSA in solution leads to the decreases in the current for the DA oxidation and the increases in the ห‫ܧ‬ଷ/ସ − ‫ܧ‬ଵ/ସ ห values at the CFEs (i.e., 55 mV) compared with that before the addition of BSA (i.e., 29 mV). These changes were attributed to the adsorption of BSA onto CFEs, which could affect the mass transport and electron transfer kinetics and inactivate the electrodes. 25,26,41-44 6

3 I / nA

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

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0

-3 -0.2

0.0 0.2 0.4 E / V vs. Ag/AgCl

0.6

Figure 2. Cyclic voltammograms (CVs) obtained at the CFE in aCSF (pH 7.4) containing 20 µM DA in the absence (black curve) and presence (red curve) of 10 mg mL-1 BSA. Scan rate, 10 mV s-1. The adsorption of proteins onto CFEs observed above is believed also to occur when CFEs are implanted in brain tissues because proteins do exist in the brain environments and may vary with the alternation of brain activity and with the electrode implantation.45-50 Such behavior may constitute a consequence for the equilibrium and variation of electrode response 10


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A

4

BSA

B BSA

I / nA

I / I0

0.9

0.6

2 0

0.3 0

300

600 900 T/s

3 C

2 I / nA

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


1200

0

400

800 T/s

1200

D Cyt. c

1 E

0

0

400

800 T/s

1200

Figure 3. (A) Amperometric responses obtained at CFEs in aCSF containing 200 µM DA upon the addition of 10 mg mL-1 BSA as indicated in the figure. Amperometric responses obtained at CFEs in aCSF containing 20 µM DA and 40 mg mL-1 BSA upon the further addition of (B) 10 mg mL-1 BSA, or (C) 5 mg mL-1 Cyt. c, as indicated in the figure. Potential applied, +0.20 V vs. Ag/AgCl. Contact angle of bare (D) and BSA-treated (E) GC electrodes.

when the CFEs are implanted in the brain. It occurs to us that the use of BSA-treated CFEs to replace bare CFEs for in vivo measurements would shorten the time employed for electrode equilibrium and minimize the influence of protein alternation caused by, for example, the change of brain activity, 48,50 on the electrode response. Our in vitro experiments suggest that BSA adsorption onto CFEs actually is a quick response (i.e., minute level), as could be seen from the current decay at CFEs evoked by the addition of BSA (Figure 3A). Therefore, the time employed for electrode equilibration could be negligible provided that the in vivo measurements are performed after the electrodes are implanted into the brain tissue for several minutes. The advantage benefited from the use of BSA-treated CFEs lies in the avoidance of the fluctuation of current response caused by the alteration of proteins around

11



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the electrodes when the electrodes are implanted into the brain tissue. As depicted in Figure 3, the further addition of BSA (B) or Cyt. c (C) into aCSF containing BSA did not lead to the further decay in the current response. Cyt. c was used here because it has a pI value of 10.2 and can interact with BSA (pI, 4.7) through electrostatic interaction. These results substantially make the electrochemical method with the BSA-treated CFEs more reliable for in vivo measurements than the existing ones with bare CFEs. Scheme 1. Schematic Illustration of Amperometric Response at the Electrode/electrolyte Interface in Different Concentration of Protein

The less fluctuation in the current response caused by further addition of proteins was understood by the surface adsorption of protein onto the BSA-treated CFEs and its effect on the electron transfer property of the electrodes. As displayed in Figure 3D, the adsorption of BSA onto electrode surface essentially increases surface hydrophilicity, which eventually provides a barrier to further adsorption of proteins onto electrode surface,51-54 regardless of the net charges of the proteins, and thereby constitutes a consequence for the less influence from the proteins on the current response at the BSA-treated CFEs, as observed in Figure 3B and C. Note that, even though the chemical environment of the brain tissue is rather complex containing many kinds of proteins with various contents, of which some proteins may over-adsorb onto the BSA-treated CFEs, the similarity in the calibrations conducted before (in aCSF containing BSA) and after (in pure aCSF) in vivo measurements substantially rules out 12


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the effect of this in vivo over-adsorption of proteins on the current response at the BSA-treated CFEs. This could be theoretically understandable (Scheme 1) when we consider the possibility that the first adsorption of BSA onto CFEs could form a densely packed protein layer, which would play a crucial role to slow down the electron transfer kinetics and inactivate the electrodes, as observed in Figure 2. The subsequent over-adsorption of proteins tends to form a loosely packed layer onto the BSA initially adsorbed onto CFEs mainly because the protein layer pre-adsorbed onto the CFEs actually increases the surface hydrophilicity and electrostatic function. The loosely packed protein layer may have less influence on the current response either in the electron transfer kinetics or the active electrode area regardless of electrochemical techniques employed such as amperometry and fast-scan voltammetry. In this sense, pre-calibration of the electrodes in aCSF containing BSA would pave a simple yet effective approach to in vivo electrochemistry with the various techniques. In order to validate the BSA-treated CFEs to monitor the release of DA, we used the BSA-treated CFE to monitor DA release in the rat NAc in vivo by bipolar stimulating in rat MFB, as displayed in Figure 4A. The released process was monitored by both amperometry (Figure 4B) and fast-scan cyclic voltammetry (FSCV) (Figure 4C). As seen in Figure 4B, the current was increased instantly ca. 2 pA after stimulation following a fast decay to baseline in 5 seconds. In order to confirm that the signal recorded by amperometry in vivo was attribute to the release of DA, FSCV was used to track DA in the process of stimulation, as typically shown in Figure 4C. By comparing with the signal recorded by amperometry, we can see stimulated DA peak pattern and kinetic recorded by amperometry and FSCV was almost the same with those reported previously.55,56 Moreover, the shape of in vivo FSCV of DA was identical to that of in vitro (Figure 4D). Note that, the pre-calibration in aCSF containing 40 mg mL-1 BSA was almost the same as the post-calibration (Figure 4E). The ratio of the sensitivity obtained with the post-calibration in pure aCSF (i.e., containing no 13



BSA) (Spost) to that obtained with pre-calibration in aCSF containing BSA (Spre) was about 94%. These results substantially validate that the BSA-treated CFE could be used for in vivo monitoring the release of DA. 5s

B

A

2 pA

100 nA

C

D

5s

50 nA 1.3 V

-0.4 V

50 nA

E 200 I / nA

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

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150 100 50 0

0.5

1.0

1.5 2.0 C / µM

2.5

Figure 4. (A) Schematic illustration of in vivo monitoring DA release in the rat NAc by stimulating rat MFB. (B) Amperometric response recorded with the BSA-treated CFE in NAc by stimulating MFB (red bar, 3 seconds at 60 Hz, ± 350 µA, 2 ms per phase). (C) In vivo FSCV recorded with the BSA-treated CFE by stimulating MFB (red bar, 3 seconds at 60 Hz, ± 350 µA, 2 ms per phase). Current versus time trace was extracted from the color plot at the peak oxidation potential (ca. +0.5 V) for DA. (D) Representative FSCV of DA in the rat NAc (red curve) and in vitro (black curve, 600 nM DA in aCSF containing 40 mg mL-1 BSA). Scan rate, 400 V s-1. (E) The calibration curves obtained in pure aCSF containing 40 mg mL-1 BSA with the bare CFEs (black curve), and in pure aCSF (i.e., containing no BSA) with the BSA-treated CFEs after in vivo implantation in NAc of rat brain for 2 h (red curve).

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BSA Pre-treated MWNT/CFEs for in Vivo Selectively Measuring Ascorbate Using Pre-calibration Method. To further validate the strategies demonstrated here for electrode calibration and use of the BSA-treated CFEs for in vivo applications, we used ascorbate as a target species because ascorbate presents a high basal level and plays important roles in the central nervous system.32,57 To achieve the selectivity for ascorbat measurement, we modified the CFEs with MWNTs to form MWNT/CFEs. The electrodes were then treated with BSA with the same procedures as those employed for DA detection described above. In brain atmosphere, ascorbate is one monovalent anion and an inner-sphere electroactive species with electron-transfer kinetics sensitive to the electrode surface. We first evaluated whether the effect of BSA on the electrochemical oxidation of ascorbate at the MWNT/CFEs. Figure 5A depicts typical CVs at the MWNT/CFE in aCSF (pH 7.4) containing 200 M ascorbate in the absence (red curve) and presence (black curve) of 10 mg mL-1 BSA. Well-defined sigmoid-shaped voltammograms (red curve) was achieved on the MWNT/CFE and the oxidation of ascorbate at this electrode reached a steady state at 0.0 V, as our reported previously.32,33 When BSA was added into ascaorbate solution, the steady-state current was clearly decreased (black curve). Furthermore, the addition of BSA induced a quick decrease in amperometric current response toward ascorbate, as shown in Figure 5B. The suppressed current was not further decreased when the concentration of BSA was continuously increased up to more than 30 mg mL-1 (shown in Figure 5C). Moreover, the addition of Cyt. c did not induce the further decay of the amperometric current response, as seen from Figure 3D. These results revealed that the effects of BSA on the current responses of ascorbate at the MWNT/CFEs were quite similar to those at the CFEs for DA described above. These results may suggest that the BSA-treated CFEs are suited for in vivo measurements of both anionic and cationic species, which was different from the electrodes coated with the traditional membranes such as Nafion. This property might result from the 15



inherent pinhole or permeability of protein coating,42,43 which enables small neurochemical molecules to diffuse through densely and loosely packed proteins to electrode.

A

B

1.0

40

BSA

0.8 I / I0

I / nA

80

0

-40

0.6 0.4

-0.2

-0.1 0.0 0.1 E / V vs. Ag/AgCl

0

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2 Cyc. c

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Figure 5. (A) CVs obtained at the MWNT/CFE in aCSF (pH 7.4) containing 200 µM ascorbate in the absence (red curve) and presence (black curve) of 10 mg mL-1 BSA. Scan rate, 10 mV s-1. (B) Amperometric current response recorded at the MWNT/CFE towards 200 µM ascorbate at +0.05 V upon the addition 10 mg mL-1 BSA as indicated in the figure. I0 and I were current values at starting time and given time, respectively. (C) Amperometric response observed at the MWNT/CFE in aCSF (pH 7.4) containing 200 µM ascorbate upon successive addition of 1 mg mL-1 BSA and subsequent 5 mg mL-1 BSA as indicated in the figure. (D) Amperometric response observed at the MWNT/CFE in aCSF (pH 7.4) containing 200 µM ascorbate and 40 mg mL-1 BSA upon addition of 5 mg mL-1 Cyt. c. Potential: +0.05 V vs. Ag/AgCl.

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8 DA DOPAC 5-HT

UA Ascorbate

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100

200 C / µM

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0 0

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Figure 6. (A) Amperometric response obtained at the MWNT/CFE in aCSF (pH 7.4) containing 40 mg ml-1 BSA upon the addition of 10 µM DA, 20 µM DOPAC, 50 µM UA, 10 µM 5-HT and 200 µM ascorbate. (B) Amperometric current responses of the MWNT/CFE toward successive addition (each addition, 50 µM) under different conditions: in pure aCSF containing 40 mg mL-1 BSA and prior to in vivo implantation (black curve), and in pure aCSF (i.e., containing no BSA) after in vivo implantation in striatum of rat brain for 2 h (red curve). Inset, calibration curves obtained under both conditions. Potential, 0.05 V vs. Ag/AgCl. (C) The ratio of sensitivity obtained pre-calibration of the MWNT/CFE in aCSF in the absence (red column) and presence (black column) of 40 mg mL-1 BSA to that of post-calibration in pure aCSF after in vivo electrode implantation for 2 h.

The BSA-adsorbed MWNT/CFEs exhibit a high selectivity against DA, DOPAC, UA and 5-HT, as shown in Figure 6A. Moreover, the calibration curve of the MWNT/CFEs for ascorbate in aCSF containing 40 mg mL-1 BSA before in vivo was almost the same as that in pure aCSF after the electrodes were subject to brain tissue implantation (Figure 6B). In comparison, the sensitivity of calibration obtained MWNT/CFE done in pure aCSF was 17



decreased significantly after in vivo measurement (Figure 6C). These results enable to selective monitor ascorbate with our developed calibration method. 10

5

I / nA

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0

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Figure 7. In vivo amperometric response recorded with the BSA-treated MWNT/CFE in rat striatum. The BSA-treated MWNT/CFE was prepared by immersing the MWNT/CFE into aCSF containing 40 mg mL-1 BSA for 1 h, taken out of the solution and washed with water. Potential applied, +0.05 V vs. Ag/AgCl.

Figure 7 depicts typical result obtained in vivo with the BSA-pretreated MWNT/CFEs. The current obtained in vivo was quite stable as a function of time. According to the pre-calibration (Figure 6B), the basal concentration of ascorbate was determined to be 205 ± 41 µM (n = 3), which was almost consistent with the reported values with other methods,32,47,57-59 further validating the strategies demonstrated here for calibrating electrodes in in vivo measurements.

CONCLUSIONS In summary, we have observed that BSA pretreatment to CFEs essentially enables in vivo electrochemical measurements with a high reliability. Our observations include, 1) the adsorption of BSA onto CFEs by immersing the electrodes in aCSF containing BSA substantially validates a new and effective pre-calibration method for in vivo measurements by adding BSA into aCSF, and 2) the use of BSA-treated CFEs as a probe for in vivo 18


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measurements virtually eliminates the variation of current responses caused by the alteration of proteins during electrode implantation and following brain activities. This study may open a new avenue to tracking brain chemistry with a high reliability and a simplified pre-calibration. ACKNOWLEDGMENT We gratefully acknowledge the financial support from National Natural Science Foundation of China (Grant Nos. 21321003, 21435007, and 21210007 for L.M. and Grant Nos. 21475149, 21522509 for M.Z.).

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Protein Pretreatment of Microelectrodes Enables in Vivo

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