In Vivo Monitoring of H2O2 with Polydopamine and Prussian Blue

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In Vivo Monitoring of H2O2 with Polydopamine and Prussian Blue-coated Microelectrode Ruixin Li, Xiaomeng Liu, Wanling Qiu, and Meining Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01765 • Publication Date (Web): 06 Jul 2016 Downloaded from http://pubs.acs.org on July 11, 2016

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Analytical Chemistry

In Vivo Monitoring of H2O2 with Polydopamine and Prussian Blue-coated Microelectrode Ruixin Li, Xiaomeng Liu, Wanling Qiu, and Meining Zhang* Department of Chemistry, Renmin University of China, Beijing 100872, China.

*Corresponding Author. Fax: +86 10 62516444; (E-Mails): [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT: In vivo monitoring peroxide hydrogen (H2O2) in brain is of importance for understanding the function of both reactive oxygen species (ROS) and its signal transmission. Robust microelectrode for in vivo measuring H2O2 is challenging due to the complexity of brain environment and the instability of the electrocatalysts employed for the reduction of H2O2. Here, we develop a new kind of microelectrodes for in vivo monitoring of H2O2, which are prepared by first electrodeposition of Prussian blue (PB) onto carbon nanotubes (CNTs) assembled carbon fiber microelectrodes (CFEs), and then over-coating of the CFEs with a thin membrane of polydopamine (PDA) through self-polymerization. Scanning electron microscopic and X-ray proton spectroscopic results confirm the formation of PDA/PB/CNT/CFEs. The PDA membrane enables PB-based electrodes to show a high stability in both in vitro and in vivo studies and to stably catalyze the electrochemical reduction of H2O2. The microelectrode is selective for in vivo measurements of H2O2 actually interference-free from O2 and other electroactive species coexisting in the brain. These properties, along with the good linearity, high biocompatibility and stability toward H2O2, substantially enable the microelectrodes to in vivo track H2O2 changes during electrical stimulation and microinfusion H2O2 and drug, which demonstrates that the microelectrode could be well competent for in vivo monitoring the dynamic changes of H2O2 in rat brain.


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INTRODUCTION More and more evidences have shown that hydrogen peroxide (H2O2) not only is toxic but also plays a crucial role functioning as both an intracellular signaling agent and a diffusible messenger like nitric oxide, in various aspects of brain function.1-10 The level of H2O2 has been demonstrated to be closely correlated with many neurodegeneration disorders such as Alzheimer’s disease and Parkinson’s disease.11-13 Therefore, in vivo monitoring of H2O2 is of great significance in both physiological and pathological investigations. In vivo electrochemistry using tissue-implantable microelectrodes is a powerful method to monitor extracellular neurochemicals with a high time and space resolution,14-21 however, in vivo detection of H2O2 with a microelectrode under physiological and pathological conditions is still a challenge because of the complexity of the cerebral environments. At most frequently used electrodes, the electron transfer kinetics of H2O2 is rather sluggish. To this end, electrocatalysis of H2O2 has been of great concern not only in the fundamental electrochemical studies but also in the practical applications such as biosensing and fuel cells as well.22-25 However, most of the catalytic processes reported so far could not apply into an in vivo electrochemical method for H2O2 detection mainly because of the poor selectivity of the methods. For instance, noble metals, such as platinum, have been employed as the electrocatalysts for H2O2; however, the reduction of H2O2 generally suffers from the interference from the dissolved O2 and the oxidation of H2O2 often bears interference from ascorbate and catecholamine accompanying with the production of hydroxyl radical.26-34 Using



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horseradish peroxidase (HRP) as biocatalyst H2O2 would form effective protocols for H2O2 biosensing through either direct or mediated electron transfer of HRP; however, these protocols are yet difficult to apply for in vivo application. For example, although the direct electron transfer property of HRP enables the as-prepared biosensors to operate at a relatively low potential (-0.3 V vs. Ag/AgCl), the biosensors normally suffer from the interference from O2.35 The biosensors based on the mediated electron transfer of HRP are subject to the interference from ascorbate through different pathways, as report previously.36 To eliminate the interference from ascorbate and thereby to enable osmium redox polymer mediated HRP biosensors for in vivo measurement of H2O2, Kulagina et al. over-coated the biosensors with ascorbate oxidase and Nafion to achieve the selectivity.25 In this study, we develop a novel microelectrode for in vivo monitoring of H2O2 with Prussian blue (PB) as an electrocatalyst to accelerate the reduction of H2O2. Due to its excellent activity toward the reduction of H2O2 at very low potential (ca. 0 V vs. Ag/AgCl), PB has been considered as an “artificial enzyme” and does not catalyze the reduction of O2,37-42 and is thus expected to be the most suitable electrocatalyst for in vivo monitoring of H2O2 in the complex cerebral environments. However, PB loses its electrocatalytic property toward the reduction of H2O2 in neutral pH because the interaction between Fe3+ and OH- frame structure results in collapse of the framework.43-45 The microelectrodes used in this study are prepared by first electrodeposition of PB at assembled carbon nanotube-modified carbon fiber microelectrodes to form PB/CNT/CFEs. The PB/CNT/CFEs are further coated with


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polydopamine (PDA) by chemical self-polymerization of dopamine to stabilize PB in in vivo neutral condition due to the three reasons. First, PDA can strongly adhere to any substrate through chemical or electrochemical polymerization,46,47 which would interact with PB. Second, PDA is considered as a zwitterionic polyelectrolyte, because of the presence of both amine groups and phenolic hydroxyl groups in its structure.48,49 PDA membrane exhibits pH-switchable permselectivity and passes cations but excludes anions at neutral pH. Third, PDA has a good biocompatibility because of its hydrophilic property.47,50 The prepared PDA/PB/CNT/CFEs exhibit a good electrocatalytic activity toward the reduction H2O2 at -0.05 vs. Ag/AgCl with a good stability and selectivity and could be used for in vivo monitoring H2O2 in rat brain.

EXPERIMENTAL SECTION Reagents and Solutions. Carbon nanotube (CNT, 0.5-50 µm and 10-30 nm in diameter) was purchased from Shenzhen Nanotech Co., Ltd (Shenzhen, China). Poly(diallyldimethylammonium chloride) (PDDA, Mw: 200 000-350 000), ascorbate (AA), dopamine (DA), uric acid (UA), 3,4-dihydroxyphenylacetic acid (DOPAC), mercaptosuccinate (MCS) and 5-hydroxytryptamine (5-HT) were purchased from Sigma and used as supplied. Other chemicals were of analytical grade at least and used as received. 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



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(1.1 mM) into double distilled water, and then adjusted to pH 7.4, which be used as the electrolyte for in vivo and in vitro electrochemical experiments. Preparation

of

Polydopamine/Prussian

Blue/CNT

Carbon

Fiber

Microelectrode. Carbon fiber microelectrode (CFE) was used as substrate electrode fabricated as reported previously.51-53 A single carbon fibre (5 µm in diameter, Tokai Carbon Co., Tokai, Japan) was carefully inserted into the capillary, pulled on a vertical pipet puller (WD-1, Sichuan, China). Prior to modification, the fabricated CFE was first sequentially sonicated in acetone, 3 M HNO3, 1.0 M KOH, and double-distilled water for 3 min. Then, the electrodes

were

subjected

to

electrochemical

activation,

first

with

potential-controlled amperometry at +2.0 V for 30 s, at -1.0 V for 10 s, and then 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. To get a stable and well-dispersed CNT, 1 mg mL-1 CNT was dispersed into aCSF containing 1 wt. % sodium dodecyl sulfate (SDS) according to our previously method.54 CNT assembled CFE was prepared by dipping the electrodes into 0.5 M NaCl containing 1 wt. % positive charged PDDA, and then negative charge CNT dispersion for 20 minutes alternatively.54 After each dipping step, the electrode was carefully rinsed with ultrapure water to remove the unassembled materials, and then dried in air. Repeat for five times, we got CNT assembled CFE (denoted as CNT/CFE).


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Prussian blue (PB) was electrodeposited at CNT/CFE by a constant cathodic current of 2 µA cm-2 for 700 s in a fresh solution containing 1 mM FeCl3 and 1 mM K3Fe(CN)6. Then the electrode was cleaned by double distilled water, dried in air and activated by applying cyclic voltammetry in 0.1 M KCl (pH=2) within a potential range from -0.2 to 0.6 V at a scan rate of 0.05 V s-1 until a stable

cyclic

voltammogram

was

obtained,

which

was

denoted

as

PB/CNT/CFE.48 To further stabilize PB, one layer of polydopamine (PDA) film was prepared through chemical method, which is immersing the electrode in aCSF containing 1 mg mL-1 DA and 1.71 mg mL-1 ammonium persulfate (molar ratio was 2:1) for 3.5 h polymerization, and then washed with double distilled water, heated at 100 °C for 2 hours, denoted as PDA/PB/CNT/CFE. Glassy carbon (GC, 3 mm in diameter) electrodes were polished with emery paper first and then with aqueous slurries of fine alumina powders (1 and 0.05 µm) on a polishing cloth, and were finally rinsed with acetone and doubly distilled

water

in

PDA/PB/CNT/GC

an

ultrasonic

bath,

each

for

was

prepared

with

the

same

several

minutes.

procedure

as

PDA/PB/CNT/CFE. Apparatus

and

Measurements.

Electrochemical

measurements

were

performed on a computer-controlled electrochemical analyzer (CHI760D, CHI Instruments, and Shanghai, China). The as-prepared PDA/PB/CNT/GC or PDA/PB/CNT/CFE was used as working electrode, and platinum wire was used as the counter electrode. A Ag/AgCl electrode (vs sat. KCl) was used as the



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reference electrode for in vitro electrochemical measurements. Scanning electron microscopy (SEM; Hitachi S4300-F microscope, Hitachi Inc., Tokyo, Japan)

was

used

for

characterization

of

the

assembled

CNT

and

electrodeposited PB onto CFE. The CNT and treated CNT was pressed into pellets for X-ray photoelectron spectroscopy (XPS) measurement. The PDA/PB/CNT was modified onto ITO substrate as the procedure as preparing the

PDA/PB/CNT/CFE

for

XPS

measurements

performed

on

an

ESCALab220i-XL electron spectrometer from VG Scientific using 300 W Al Kα radiations. Zeta potential was measured using a Malvern Zetasizer Nano ZS90. Electric stimulation was generated using an isolator (ISO-Flex, AMPI, Israel). In Vivo Experiments. In vivo experiment was performed as previously reported. Briefly, experiments involving adult male Sprague-Dawley rats (250-300g) were purchased from Centre for Health Science, Peking University. All in vivo experiment 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 earlier work.52,55 After housed on a 12:12 h light-dark schedule with food and water as libitum, the rats were anaesthetized with chloral hydrate (345g/kg, ip) and fixed onto a stereotaxic frame as our previously report.55,56 By using stereotaxic procedures, the PDA/PB/CNT/CFE was inserted into the right cortex (AP = 3


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mm, L = 2 mm lateral from bregma, V = 1 mm from the surface of the skull). The prepared microsized Ag/AgCl reference electrode was positioned into the dura of brain. Platinum wire was used as the counter electrode inserted in subcutaneous tissue on the brain. Exogenous aCSF containing 100 µM H2O2 or MCS were microinfused to the local area of implanted microelectrode in the brain through silica capillary tube (4 cm length, 50 µm i.d., 375 µm o.d.). The capillary tube was implanted into the right cortex parallel combined with PDA/PB/CNT/CFE. These solutions were delivered from gas impermeable syringes and pumped through tetrafluoroethylenehexafluoropropene (FEP) tubing by a microinjection pump (CMA100, CMA Microdialysis AB, Stockholm, Sweden). All local microinfusions were performed in the striatum of the rat brain at 2.0 µL min-1. A bipolar stimulating electrode was implanted in the right cortex combined with PDA/PB/CNT/CFE, and a stimulation of 10 s at 5 V was applied.

RESULTS AND DISCUSSION Characterization and Property of Polydopamine/Prussian Blue/Carbon Nanotube/Carbon Fiber Microelectrode (PDA/PB/CNT/CFE). CNT, considered as their unique electronic conductive and chemical properties, is used to construct 3D conducting framework and act as wire to electrocatalytically, therefore, efficiently enlarges the active surface area and enhances sensitivity in electrochemical applications.57-60 Nevertheless, typical methods of modifying CNT onto CFE, such as



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dipping-coating and drop-coating, often suffer from non-uniform. Layer-by-layer is a suitable method to form uniform film of CNT onto substrate by self-assembling negatively charged CNT, and positively charged PDDA through electrostatic interaction.54 Figure 1 shows scanning electron microscopy (SEM) images of bare CFE (A), assembled CNT/CFE (B), and PB deposited on CNT/CFE (C, D), respectively. From Figure 1 we can clearly see that compared with the bare CFE, CNT distributed on the surface of CFE uniformly. PB nanoparticles were captured in the CNT network. CNT could not only construct 3D conducting framework, where electron exchange occurs between all the nanoparticles and the electrode because of its excellent electronic conductivity, but also increases the effective surface area of electrode and the loading amount of PB.43,58 These properties are benefit from the inherent

advantages

of

CNT,

which

would

enhance

the

sensitivity

of

PB/CNT/CFE.57-60 PDA film, oxidant-induced DA polymerization, can be considered as an excellent zwitterionic polyelectrolyte and exhibits permselectivity for cations and anions at different pH because of containing phenolic hydroxyl groups and amine groups in PDA’s structure, as reported previously.47,48,50,61-66 Taking Fe(CN)63- and Ru(NH3)63+ as anion and cation electrochemical probe, we investigated their electrochemical response at PDA-modified electrode in aCSF (pH 7.4), the results are shown in Figure 2. As shown, compared the current responses obtained at bare GC (black curve), electrochemical response of both Fe(CN)63- and Ru(NH3)63+ at PDA/GC electrode (red curve) were remarkably decreased, and even Fe(CN)63- almost had no current


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response at PDA/GC electrode. Meanwhile, the zeta potential of PDA particles was -0.163 V in neutral solution. These results indicate that PDA film could exclude ions regardless of charge state, especially molecule with negative charged because of negative impulsion. In contrast, non-charged H2O2 had the same electrochemical response at both bare electrode (black curve) and PDA-modified Pt electrode (red curve), as shown in Figure 2C. These results demonstrate that the PDA membrane would avoid anions entering the membrane, such as OH-, AA, while not affecting the detection of H2O2. This property substantial benefits for stability of PB and the good selectivity of PDA/PB/CNT/CFE. PB has a basic cubic structure consisting of alternating iron (II) and iron (III) located on a face centered cubic lattice. In such a way, the iron (III) ions are surrounded octahedrically by nitrogen atoms, and iron (II) ions are surrounded by carbon atoms.45 Figure 3 shows the typical XPS spectra of (A) Fe 2p and (B) N 1s of PB (black curve), PDA (red curve), and PB/PDA (blue curve). The XPS spectra of PB exhibited two Fe 2p peaks at 708 eV and 721 eV, and one N 1s peak at 397.5 eV.67 The spectra of PDA showed one N 1s at 399.5 eV.68 In XPS spectra of PDA/PB membrane, we can observe peaks both of Fe 2p and N 1s. This result indicates that PDA and PB were confined onto the substrate as our procedure successfully. On the other way, the band energies of Fe 2p and N 1s of PDA/PB shifted ca. 1 eV in comparison with those of PB, which suggests that there is some interaction between PB and PDA. This interaction would benefit to enhance stability of PB in neutral solution.



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PB is an artificial enzyme of H2O2, which could electrocatalize H2O2 reduction at low potential (ca. 0 V vs Ag/AgCl). This low potential could benefit for minimizing the interference of other electroactive molecules, such as DA. However, the challenge of PB applied in in vivo monitoring is the stability in the brain atmosphere because PB has poor stability in neutral media due to the strong interactions between high spin Fe3+ and OH-, hence resulting in collapse the polycrystalline structure of PB nanocrystal.39,44,45,69 Figure 4 demonstrates the stability of PB at different CFE. Figure 4A shows the typical CV obtained at PB/CNT/CFE. There was one pair of redox peaks at ca. +0.1 V, which is attributed to the oxidation and reduction process of PB.39,67,70 The current response obtained at PB/CNT/CFE (Figure 4A) decreased with increasing the cycle number in aCSF (pH 7.4) at a scan rate of 50 mV s-1. Although PB/CNT/CFE could improve the stability of PB to some extent compared to that obtained at PB/CFE, the current of PB was decreased slowly. In comparison, the current response of PDA/PB/CNT/CFE did not decrease after 50 cycles in the same condition (Figure 4B). Moreover, this good stability remains in brain atmosphere (Figure 4C). Such an excellent stability might be ascribed to two reasons of PDA membrane. The first is the interaction of PDA and PB resulted in stable crystal structure of PB. The second is the ion permselectivity of PDA film. Negative charge of PDA membrane at neutral solution impedes OH- to access to PB, then prevents PB solubilizing and improves the stability of PB. Meanwhile, Figure 4D shows typical CVs


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observed at PDA/PB/CNT/CFE in a CSF (pH 7.4) with the absence (red curve) and presence (black curve) of 0.2 mM H2O2. With the addition of H2O2, the value of oxidation peak current decreased but reduction one increased, which demonstrates the PDA/PB/CNT/CFE has good electrocatalytic property towards the reduction of H2O2.

Linearity, Selectivity, Stability of PDA/PB/CNT/CFE toward the Reduction of H2O2. In the cerebral system, the O2 level varies with brain regions (striatum, hippocampus, etc.) and would fluctuate involved in almost physiological and pathological processes.71,72 One great challenge of H2O2 microprobe is how to avoid the interference from O2. Therefore, choosing appropriate potential and electrocatalyst are key issues to improve the sensitivity and selectivity of the electrochemical biosensors. As shown is Figure 5, at -0.05 V, compared with 10 µM H2O2, no obvious current response of 12.5 µM O2 was obtained at PDA/PB/CNT/CFE, even though successive addition of 6 times.55,73 On the contrary, at -0.1 V 10% cathodic current of 12.5 µM O2 was obtained, compared with 10 µM H2O2, which might be attributed to the electrocatalytic reduction of O2 at CNT. This result indicates that, compared with a more negative potential, for example, at -0.1 V, the sensitivity and selectivity of PDA/PB/CNT/CFE increased, and the interference from O2 could be ignored when applied potential at -0.05 V. Therefore, -0.05 V was selected as

the

potential

for

fulfilling

sensitivity

and

selectivity

of

PDA/PB/CNT/CFE for in vivo tracking H2O2 dynamic in the presence of O2.


the


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Plenty of endogenous electroactive species including cations and anions such as AA, DA, DOPAC, UA, and 5-HT common coexisting in the rat brain could be readily electrochemically oxidized at the microelectrode and may thus potentially interfere with the in vivo monitoring of H2O2. No obvious response of coexisting electroactive species (200 µM AA, 5 µM DA, 50 µM DOPAC, or 5 µM UA) was observed compared with the large response of 5 µM H2O2, as shown

in

Figure

6A,

highlighting

the

remarkable

specificity

of

PDA/PB/CNT/CFE. The stability of PDA/PB/CNT/CFE towards continuous monitoring H2O2 was also studied in vitro. As note, AA is oxidized at -0.05 V at CNT/CFE, as reported previously.52 While AA has no electrochemical response at PDA/PB/CNT/CFE, which might be attribute to permselectivity of PDA film at the outside of present CFE. The result (Figure 6B) shows that the amperometric response obtained at PDA/PB/CNT/CFE remained stable for almost 1 hour. Figure 7 displays typical current response at the PDA/PB/CNT/CFE upon the successive addition of H2O2 in aCSF at -0.05 V vs. Ag/AgCl. The as-prepared PDA/PB/CNT/CFE exhibited excellent catalytic activity toward H2O2 reduction and well amperometric response upon successive addition of H2O2. The amperometric response was linear with the concentration of H2O2 from 1 µM to 140 µM (I (nA) = 2.71 (nA) + 0.094CH2O2 (µM), R = 0.99). The detection limit was 0.4 µM (S/N = 3).


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It is worth noting that the outer layer of PDA/PB/CNT/CFE is PDA, which is zwitterionic polyelectrolyte. This property will increase the hydrophilicity of microprobe,

and

hence

improve

the

biocompatibility

of

implanted

microelectrode.47,74,75 This assumption is confirmed by the results that the contact angle of PDA coated electrode (Figure 8A) was smaller than that of bare electrode (Figure 8B) and the amperometric response was almost same obtained at before (red) and after (black) immersing PDA/PB/CNT/CFE into 10 mg mL-1 BSA (Figure 8C). These properties combined with good linearity of PDA/PB/CNT/CFE would be benefit to well responding in in vivo study. In Vivo Monitoring H2O2 with PDA/PB/CNT/CFE. Figure 9 shows amperometric response of PDA/PB/CNT/CFE implanted in the cortex of anesthetized rat when local microinfusion of aCSF (red curve), aCSF containing

100

µM

H2O2

(black

curve)

(A),

aCSF

containing

mercaptosuccinate (MCS) (B) and electrical stimulation (C) in the near of PDA/PB/CNT/CFE. As shown in Figure 9A, a large and fast increased current response was observed while exogenously infused 100 µM H2O2 and the increased current returned to the baseline after stop infusion. On the contrary, there was no current change obtained at PDA/PB/CNT/CFE while injection of pure aCSF. These results indicate that the increased current when infusion was related to H2O2. MCS could amplify endogenous levels of H2O2 through inhibiting the activity of GSH peroxidase, as well studied by Rice and others.6,76,77 As shown in Figure 9B, locally microinfused MCS caused an



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evident, delayed increase in extracellular H2O2 concentration (Figure 9B). And the large concentration of MCS induced large increase of H2O2 level. This phenomenon of H2O2 fluctuation during infusion of H2O2 and MCS is same with the reported literature,6 which demonstrate our microelectrode could in vivo track the changes of H2O2 in physiological and pathological process associated with H2O2. Because the developed microelectrode has good selectivity in sensing H2O2 without interference of O2, it enables to monitor the dynamic changes of H2O2 in pathological and physiological process associated with the level changes of O2, such as electrical stimulation.25 Figure 9C shows that the amperometric current response decreased a little after electrical stimulation firstly, then increased, and finally returned back to baseline after ca. 2 min. It is reported that electrical stimulation could induce the change of neutral activity and the regional cerebral blood flow, which might be attributed to the changes of H2O2 in the process of electrical stimulation. 78

CONCLUSIONS In summary, we successful prepared one robust H2O2 microprobe for in vivo tracking the changes of H2O2 using PB as electrocatalyst coated by PDA membrane. The developed PDA/PB/CNT/CFE had good stability in vivo and in vitro, good electrocatalytic response towards continuous monitoring H2O2 with enough selectivity, and could be used in in vivo monitoring H2O2 dynamic


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during infusion of H2O2 and drug, and electrical stimulation. These results demonstrate the developed microprobe is one good candidate for in vivo monitoring H2O2 to understand the function of H2O2 in brain chemistry. Moreover, the developed microelectrode could be used for fabricating oxidase-based microprobe for in vivo measuring other species, such as glutamate and glucose.

ACKNOWLEDGEMENTS We gratefully acknowledge the financial support from National Natural Science Foundation of China (Grant Nos. 21475149 and 21522509).

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Scheme 1

Scheme 1. Scheme of the Microbiosensor Implanted in Rat Brain and the Structure of PDA/PB/CNT/CFE.

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Figure 1

Figure 1. Scanning electron microscopy (SEM) images of (A) bare CF, (B) CNT/CFE, and (C, D) PB/CNT/CFE.

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Figure 2

60

A

60

B

40 C

0

-60

-30 -60 -0.3

0

I / µA

I / µA

30

I / µA

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0.0

0.3

0.6

E / V vs. Ag/AgCl

-120

20 0

-0.4

-0.2

0.0

0.2

E / V vs. Ag/AgCl

-20

0.0

0.4

0.8

E / V vs. Ag/AgCl

Figure 2. Typical cycle voltammograms (CVs) obtained at PDA/GC (red curve) and bare GC (black curve) in aCSF (pH 7.4) containing (A) 1 mM Fe(CN)63- and (B) 1 mM Ru(NH3)63+. (C) Typical CVs obtained at PDA/Pt (red curve) and bare Pt (black curve) in aCSF containing 0.2 mM H2O2. Scan rate, 50 mV s-1.

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Figure 3

B

A

700

710 720 730 740 Band Energy (eV)

390

395 400 405 410 Band Energy (eV)

415

Figure 3. XPS spectra for (A) Fe 2p and (B) N 1s of PB (black curve), PDA (red curve), PB/PDA (blue curve).

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Figure 4

A

B

0.4 I / µA

I / µA

0.3

0.0

0.0

-0.4

-0.3 -0.3

-0.3

0.0 0.3 0.6 E / V vs. Ag/AgCl

0.5 C

0.06

0.0 0.3 0.6 E / V vs. Ag/AgCl

D

I / µA

0.00

I / µA

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0.0

-0.06

-0.5 -0.3

Figure

4.

Typical

-0.12

0.0 0.3 0.6 E /V vs. Ag/AgCl

consecutive

CVs

-0.2

obtained

0.0 0.2 0.4 0.6 E /V vs. Ag/AgCl

at

(A)

PB/CNT/CFE

and

(B)

PDA/PB/CNT/CFE in aCSF (pH 7.4). (C) CVs obtained at PDA/PB/CNT/CFE in rat cortex. Scan rate, 50 mV s-1. (D) CV obtained at PDA/PB/CNT/CFE in aCSF without (red line) and with 0.2 mM H2O2 (black line). Scan rate, 10 mV s-1.

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Figure 5

A

T/s B

T/s Figure 5. Amperometric response of successive addition O2 and H2O2 at PDA/PB/CNT/CFE (vs. Ag/AgCl) in the N2-atmosphere aCSF. The concentration for each O2 and H2O2 is 12.5 µM and 10 µM. Potential applied: (A) -0.1 V and (B) -0.05 V.

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Figure 6

A

T/s B

T/s Figure 6. Amperometric response obtained at PDA/PB/CNT/CFE microelectrode in aCSF (A) with successive addition of 200 µM AA, 20 µM DA, 10 µM DOPAC, 50 µM UA, 10 µM 5-HT, 5 µM H2O2, and (B) upon the addition of 5 µM H2O2 as indicated. The electrode was polarized at -0.05 V vs. Ag/AgCl.

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Figure 7

T/s Figure 7. Amperometric response curve obtained at the PDA/PB/CNT/CFE in the aCSF upon successive addition of different concentration of H2O2 labelled in the figure. Applied potential: -0.05 V vs. Ag/AgCl.

29



Figure 8

B

A

C 4

∆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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2

2

4

6

CH2O2 / µM

8

Figure 8. The contact angle of (A) bare GC and (B) PDA/GC electrode. (C) Amperometric response of different concentration of H2O2 obtained at PDA/CNT/PB/CFE before (black) and after (red) immersed in aCSF containing 10 mg mL-1 BSA for 3 h.

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Figure 9

A

2 nA aCSF H2O2

0

300

600

TT/ s/ s

900

20 nA

B 1 mM MCS 10 mM MCS

T/s C

0

1 nA

200

400

TT/ /ss

600

Figure 9. Amperometric response recorded at PDA/PB/CNT/CFE in the cortex of the anesthetized rats (A) during local microinfusing of pure aCSF (red curve) and aCSF containing 100 µM H2O2 (black curve) (B) 1 mM MCS and 10 mM MCS indicated in the Figure, and (C) during electrical stimulation (5 V, 5 s). The electrode was polarized at -0.05 V vs. Ag/AgCl.

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In Vivo Monitoring of H2O2 with Polydopamine and Prussian Blue

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