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An Electrochemical Microsensor for theDetection of Nitric OxideDongyun Zheng a , Xiaojun Liu a , Huimin Cao a , Shanying Zhu a &Yaguang Chen aa College of Biomedical Engineering, South-Central University forNationalities , Wuhan , ChinaAccepted author version posted online: 31 Oct 2012.Publishedonline: 01 Mar 2013.

To cite this article: Dongyun Zheng , Xiaojun Liu , Huimin Cao , Shanying Zhu & Yaguang Chen (2013)An Electrochemical Microsensor for the Detection of Nitric Oxide, Analytical Letters, 46:5, 790-802,DOI: 10.1080/00032719.2012.738348

To link to this article: http://dx.doi.org/10.1080/00032719.2012.738348

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Sensors

AN ELECTROCHEMICAL MICROSENSOR FORTHE DETECTION OF NITRIC OXIDE

Dongyun Zheng, Xiaojun Liu, Huimin Cao, Shanying Zhu, andYaguang ChenCollege of Biomedical Engineering, South-Central University forNationalities, Wuhan, China

An electrochemical microsensor has been developed for the detection of nitric oxide.

The microsensor was fabricated based on a novel nanocomposite film that has good electro-

catalytic effect to the oxidation of nitric oxide. Electrochemical techniques, infrared

spectroscopy, and scanning electron microscopy were used to characterize the nano-

composite film and the electrochemical behavior of nitric oxide were investigated. The

microsensor was successfully used for the determination of nitric oxide in standard solutions

from 1.8� 10�8mol/L–7.6� 10�5mol/L, with a low detection limit of 5 nmol/L (S/N¼ 3).

The advantages of the proposed microsensor include simplicity, short analysis time, cost

effectiveness, and selectivity. The results demonstrate the feasibility of a nitric oxide assay

in practical sample analysis.

Keywords: Electrochemistry; Microsensor; Nitric oxide

INTRODUCTION

Nitric oxide (NO) is a simple small molecule and plays many important roles inorganisms (Cury et al. 2011; S. F. Kim 2011; Dhir and Kulkarni 2011); moreover,whether it can play its important role is closely related to its concentration. There-fore, to realize the sensitive, accurate, and real-time detection of nitric oxide (NO)is of great significance for biochemistry, medicine, and pharmacology. Comparedwith other detection methods, electrochemical sensing method attracts more atten-tion because of its simple operation, real-time detection, fast response, high sensi-tivity, and high anti-interference ability (Privett, Shin, and Schoenfisch 2010).These advantages contribute to promote the use of electrochemical sensing methods

Received 1 September 2012; accepted 1 October 2012.

This research is supported by the National Nature Science Foundation of China (Nos. 31070885;

61178087), the National Nature Science Foundation of Hubei Province of China (No. BZY09010), the

Scientific Research Team Project of South-central University for Nationalities (No. XTZ09002), and

the Fundamental Research Funds for the Central Universities (Nos. CZQ10003; CTZ12001; CZQ12013).

Address correspondence to Xiaojun Liu, College of Biomedical Engineering, South-Central

University for Nationalities, Wuhan 430074, China. E-mail: toliuxiaojun@hotmail.com

Analytical Letters, 46: 790–802, 2013

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032719.2012.738348

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in vivo NO determination. In order to develop a suitable sensor for in vivo and insitu applications, the size of the electrode should be fully considered. The electrodeshould be miniaturized enough to match the small space in biological micro-environments, such as single cells or certain locations in tissues. Although manyNO electrochemical sensors with high properties have been reported (Fei, Hu, andShiu 2011; F. Wang et al. 2011; Deng, Wang, and Chen 2010; Kannan and John2010; Peng et al. 2009; Sivanesan and John 2010), most of them cannot be appliedin single cell analysis and in vivo analysis because of their big dimension. Thereare only a few reports on the applications of NO microsensors in vivo analysis(Finnerty et al. 2012; Griveau et al. 2009; Du et al. 2008; Mas, Escrig, andGonzalez-Mora 2002; Mao et al. 1998); therefore, it is necessary to try our best todevelop more and more sensitive and selective NO microsensors to realize the scathe-less in vivo determination.

Carbon materials, especially the new types of carbon structures, nanofibers(CNFs) and nanotubes (CNTs), have attracted considerable attention due to theirunique mechanical and electronic properties. For example, one of the most impor-tant applications is related to their use as electrode modifier in chemical sensorsand biosensors (Li, Hoa, and Kim 2010; Tang et al. 2011). Moreover, compared withCNTs, CNFs not only have more disordered structure and more active sites onthe outer wall (Kim and Lee 2004; Werner et al. 2005), but also have better disper-sibility and wettability (Salimi, Compton, and Hallaj 2004). The higher ratio ofdefect sites, the faster of electron transmitting rate will be (Banks and Compton2005). However, electrochemical inertness and low chemical reactivity of raw CNFsand CNTs lead to the basal limitations of their applications (Dongil et al. 2011).Therefore, methods for functionalizing and processing CNFs and CNTscan improve their operability including their modification on electrode surface tofabricate electrochemical sensors. Recently, several methods have been reportedfor the functionalization of CNFs and CNTs (Mapkar, Iyer, and Coleman 2009;Li and Coleman 2008; Barroso-Bujans et al. 2007; Wang and Lin 2008). Theobtained functionalized nanocomposites preserve the unique properties of nano-fibers, simultaneously endowing the materials with novel functions that cannototherwise be acquired by raw CNFs. In this work, we functionalized CNFs andCNTs through a simple non-covalent method successfully, which not only improvedthe operability of CNFs and CNTs but also did not destroy their ontic structures.

CNFs can be modified on electrode surface through different ways, such aswith the aid of conduction paste, chemical vapor deposition, dripping and so on,however, these methods are not suitable for microelectrode and irregular electrodemodification, so looking for simple and controllable methods for modifying CNFson microelectrode surface is essential to the development of microsensors.

In this work, we functionalized CNFs and CNTs non-covalently and dispersedthem in alizarin red (AR) aqueous solutions, and immobilized them on the surface ofcarbon fiber electrodes (CFEs) via a simple and well-controlled in situ electro-polymerization method. Electrocatalytic ability of CNFs and CNTs to the oxidationof NO was compared and CNFs show better effect. The resulting PAR-CNF com-posite-modified CFEs (PAR-CNF=CFEs) were characterized with several techniques.These characterizations revealed that PAR-CNF composite formed a uniform thinfilm with a porous nanostructure and a large surface area on the CFE. This electrode

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exhibited a sensitive electrochemical response for the oxidation of NO, a property thatwas successfully applied to the detection of NO released frommacrophages. Comparedwith other NO electrochemical microsensors based on CFEs (Malinski et al. 1993; Parket al. 1998; Friedemann, Robinson, and Gerhardt 1996; Wang, Li, and Hu 2005; Duet al. 2008; Peng et al. 2008), this NO microsensor has similar or better performances(Table 1). The present work not only provides a sensitive electrochemical method forthe detection of NO in biomedical samples, but also foresees the promising applicationsof noncovalent adsorption of conjugated aromatic compounds on CNFs for the devel-opment of high-performance electrochemical microsensors.

EXPERIMENTAL

Chemicals and Apparatus

CNFs (l¼ 310 mm, U¼ 80–120 nm) were supplied by World Precision Instru-ment Inc. (WPI). MWNTs (purity >90%, U> 50 nm) were purchased from Nano-times Co. (Chengdu, China). Carbon fibers (U¼ 7.8 mm) were purchased fromKureha Chemical Industries Co., Ltd (Tokyo, Japan). AR, ascorbic acid (AA),and uric acid (UA) were purchased from China Pharmaceutical Group ShanghaiChemical Reagent Company. Dopamine (DA) was purchased from Fluka.L-arginine (L-Arg) and NG-nitro-L-arginine (L-NNA) was purchased from RujiBiological Technology Development Co., Ltd (Shanghai, China). 1% Nafion wasobtained through diluting 5% Nafion (Sigma) with alcohol. Macrophage solutionwith a concentration of 1.0� 106 cells=mL was supplied by School of Public Healthof Wuhan University. Aqueous solutions were prepared with double redistilledwater. High purity nitrogen gas was used for deoxidization.

NO-saturated solution was prepared as described previously (Friedemann et al.1996), using a value of 1.8mmol=L (mM) for its concentration at saturation (Butlerand Williams 1993). The NO gas was generated by slowly dropping a 6M H2SO4

solution into a rockered flask containing saturated NaNO2 solution; NO beingformed in this disproportional reaction. The gas generated was forced to bubblein 30% NaOH solution twice and in water once in order to trap any NO2 formedas a result of oxidation of NO from traces of oxygen. Before the addition ofH2SO4, all apparatus were degassed meticulously with nitrogen for 30min to exclude

Table 1. Performance comparison of NO electrochemical microsensors based on modified CFE

Modifiers

Linear

range (mM)

Detection

limits (nM)

Response

time References

Ni-porphyrin=Nafion No report 10 10ms Malinski et al. 1993

Nafion=m-dihydroxybenzene 0–10 60–80 5 s Park et al. 1998

Nafion=ortho-

phenylenediamine

0.1–6 35 100ms Friedemann et al. 1996

MWNTs=Nafion 0.2–86 20 No report Y. Z. Wang et al. 2005

SWNTs=Nafion 0.45–3.6 4.3 125ms Du et al. 2008

Poly(bromophenol blue) 0.036–89 3.6 30 s Peng et al. 2008

PAR-CNF 0.018–76 5 2 s Our work

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O2, as NO is rapidly destroyed by O2. NO-saturated solution was obtained bybubbling NO pure gas through 6mL deoxygenated distilled water for 30min andkept under NO atmosphere until use. NO gas is toxic at concentration higher than100 ppm, so the bubbling procedure was carried out in a fume hood. NO standardsolutions were prepared by making serial dilutions of the saturated NO solutionas previously reported (Trevin, Bedioui, and Devynck 1996). NO solutions weremade fresh and kept in a glass flask with a rubber plug, stored in a light-free place.

All electrochemical measurements were performed on a CHI 660A electro-chemical analyzer and a weak current amplifier (Shanghai Chenhua Co., China) ina conventional three-electrode arrangement, equipped with a platinum wire counterelectrode, a saturated calomel electrode (SCE) reference electrode, and a Nafion=PAR-CNF=CFE working electrode. For the deoxygenated experiments, the electro-lyte was bubbled with high-purity nitrogen for 15min and maintained nitrogen con-dition during the experiments. Fourier Transform (FT) IR spectra were measured ona Nicolet Magna-IR 550 spectrometer in reflection mode. Scanning electronmicroscopy (SEM) was performed on a scanning electron microscope (Quanta200, Fei, Holland). An inverted microscope (XDP-1) was used to control the lengthof the CFE.

Preparation of CNF-AR and CNT-AR Dispersion

The oxidation treatment of CNFs with nitric acid not only does not destroytheir original structure but also can fabricate a large number of oxygen containinggroups on CNFs (Maldonado and Stevenson 2004). These oxygen containing groupsplay important roles in improving the electrocatalytic ability of CNFs (Musamehet al. 2002). CNF-AR dispersion was prepared as follows: (1) 20mg CNFs wereadded into 40mL solution composed of 30mL concentrated H2SO4 and 10mLconcentrated HNO3, and sonicated for 4 h; (2) The mixture was diluted to 200mLand filtered with the aid of polytetrafluoroethylene filter disc of 0.22 mm pore size,then it was washed with water thoroughly until the filtrate was neutral; (3) the filtercake was dried in thermostat drying box at 50�C and the obtained powder wasoxidated CNFs; and (4) 10mg oxidated CNFs were added into 10mL 5mg=mLAR solution. After sonicating for 6 h, the CNF-AR dispersion was obtained.

For comparison, CNT-AR dispersion was also prepared through a similarway by replacing CNFs with CNTs.

Preparation of Modified Electrodes

CFEs were prepared according to a method modified from our previous work(Wang et al. 2005). Scheme 1 shows the structure of CFEs. To ensure all the CFEshave the same effective surface area, the obtained CFEs were pre-elected throughsweeping from �0.2 to 0.8V in 0.1M KCl containing 2.0mM K3[Fe(CN)6]. Priorto surface modification, the CFEs were pretreated by sweeping from �0.8 to 1.0Vin 0.1M H2SO4 for 20 cycles by cyclic voltammetry.

The fabrication of Nafion=PAR-CNF=CFE denoting as NO microsensor wascarried out as follows: (1) Through sweeping the CFE in the range of 0.0V–2.2V ata scan rate of 50mV=s for 25 cycles in CNF-AR dispersion without any supporting

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electrolyte, the PAR-CNF=CFE was obtained; and (2) the Nafion=PAR-CNF=CFE was fabricated by dipping PAR-CNF=CFE in 1% Nafion for five times, eachfor 10 s. Here, Nafion was used to improve the selectivity and anti-inactivationability of the NO microsensor. The fabrication processes of Nafion=PAR-CNF=CFE are generalized in Fig. 1.

For comparison, a Nafion=PAR-CNT=CFE was also fabricated througha similar way by replacing CNF-AR dispersion with CNT-AR dispersion.

PAR-modified CFE (PAR=CFE) was prepared by scanning a CFE in aqueousAR solution (5mg=mL) with cyclic voltammetry in the range of 0.0V–2.2V at a scanrate of 50mV=s for 25 cycles. To obtain the FTIR characterizations of AR and PAR,a PAR=GCE was prepared through a similar operation and a light blue film wasobserved on the electrode surface of GCE during this process.

Scheme 1. Scheme of carbon fiber microelectrode.

Figure 1. Fabrication of NO microsensor and detection of NO released from macrophages. (Figure

available in color online.)

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Prior to use, all of the aforementioned modified electrodes were firstwashed with water and then swept in the potential range of �0.2V to 1.2V in0.1M phosphate buffer solution (PBS, pH 7.4) at a scan rate of 50mV=s until stablecyclic voltammograms were obtained.

RESULTS AND DISCUSSION

Characterizations of CNF-AR

CNF is a kind of one-dimensional carbon nanomaterial with sp2 hybridization(Henning and Salama 1998), so it has a large number of p-electrons, which makesthey can integrate with other p-electrons with the aid of p-stacking (Otobe et al.2002). Original CNFs are hydrophobic, and oxidation treatment can bring lots ofpolar oxygen-contained groups including carboxyl and carbonyl to the surface ofCNFs, which can improve the hydrophilicity of CNFs greatly (Li, Yao, and Liang2003). ARs are a kind of organic dyes with rigid conjugated structure having largenumbers of p-electrons; moreover, ARs are amphiphatic molecules having bothhydrophilic groups (sulfonic group, hydroxyl group, carbonyl) and hydrophobicgroups (benzene ring). Therefore, AR can interact with CNFs through both Vander Waals force and p-stacking function. This interaction can make CNFs bring alot of hydrophilic groups, which allows them to dissolve in water well (Wu andHu 2004). Wu and Hu (2004) had functionalized CNTs non-covalently with ARbased on the same mechanism and dispersed CNTs in water stably.

The cyclic voltammograms for the surface modification of CFEs by PAR-CNFcomposite films via electropolymerization are shown in Fig. 2. The oxidation peakcurrent of the AR monomer at about 0.8V decreases with an increasing number

Figure 2. The in situ electropolymerization process of AR and CNFs on the surface of CFE. Scan rate:

50mV=s.

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of electropolymerization cycles (insert of Fig. 2), demonstrating the successful depo-sition of PAR films on the CFE. SEM characterization can ensure this conclusion.

Figure 3 shows the spectra of the AR monomer and PAR that were obtainedon a glassy carbon slice. Obvious differences could be observed from the spectra ofthe AR monomer (thin line) and PAR (thick line). The absorptions associated withthe aromatic C=C stretching vibration (1450 cm�1, 1595 cm�1) and the out-of-planebending vibration (1670 cm�1, 1814 cm�1, 1976 cm�1, 2153 cm�1) barely changedfor these two substances. However, there is one major difference between the spectraof the AR monomer and PAR: the absorption associated with the phenol C-Ostretching vibrations (1200 cm�1) is almost absence in the spectrum of AR monomer,but is broad and weak in the spectrum of PAR, indicating the possible enhancementof phenol C-O structure during the electropolymerization of AR monomers.

SEM was used to characterize the surface morphologies of bare CFEs, PAR=CFEs and PAR-CNF=CFEs and Fig. 4 shows the results. The surface of bare CFEis rather smooth (Fig. 4A), and the surface modification by PAR produces a thin butnonuniform planar film on CFEs (Fig. 4C). In contrast, the PAR-CNF compositefilm on CFEs possesses a three-dimensional and porous structure with lots ofwell-dispersed CNFs (Fig. 4B), which can significantly increase the surface area ofCFEs and may allow the free entry of analytes into the inner layers, increasing uti-lization of the whole film.

Detection of NO with Nafion/PAR-CNF/CFE

Figure 5 shows the square wave voltammograms of different electrodes in0.1M deaerated phosphate buffer solution (pH 7.4). For 18 mM NO, a small, wideoxidation peak at 1.1V with a current of 17.3 nA is observed (curve b) on the bareCFE. The oxidation current increased to 37.1 nA and 131.9 nA on Nafion=PAR=CFE (curve c) and Nafion=PAR-CNT=CFE (curve d), respectively, the peak also

Figure 3. Infrared spectra of AR (thin line) and PAR (thick line).

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exhibits a negative shift of the oxidation potential to 0.8V and 0.77V, respectively.However, a sharp oxidation peak at 0.74V with a current of 273.6 nA is also presentwith the Nafion=PAR-CNF=CFE (curve e), when the concentration of NO is addedto 36 mM, the peak current increased to 353.4 nA (curve f). There is no electrochemi-cal signal in the absence of NO (curve a). These results suggest that the observedelectrochemical response belongs to the electrochemical oxidation of NO and thePAR-CNF film can catalyze the electrochemical oxidation of NO on CFEs. Thiseffect should arise from the synergetic enhancement of PAR and CNFs: CNFs havelarge specific surface area, strong adsorption ability and good conductivity, which is

Figure 4. SEM images of bare CFE (A), PAR-CNFs=CFE (B), and PAR=CFE (C).

Figure 5. Square wave voltammagrams of bare CFE (curve b), Nafion=PAR=CFE (c), Nafion=PAR-

MWNT=CFE (d), and Nafion=PAR-CNF=CFE (a, e, and f) in the absence of NO (a), and in the

presence of 18 mM NO (b, c, d, and e) and 36mM NO (f) in 0.1M deaerated phosphate buffer solution

(pH 7.4). Amplitude: 50mV. Frequency: 15Hz.

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favorable toward the enrichment of NO and can accelerate the electron transferbetween NO and electrode; PAR is an oligomer which can decrease proton-givingability and increase the electrocatalytic ability.

We performed amperometric measurements in 0.1M deaerated phosphatebuffer solution (pH 7.4) at an operation potential of 0.8V under stirring. The resultsare shown in Fig. 6. The amperometric response of different concentrations of NOon the microsensor was rapid (�2 s) and had excellent reproducibility (Fig. 6Aand Fig. 6B). The current response exhibited a linear relationship with the con-centration of NO in the range of 18 nM–76 mM, Ip (nA)¼ 1.51þ 2.77c (mM),R¼ 0.999. The sensitivity was 2.77 nA=mM. The detection limit was detected tobe 5 nM (S=N¼ 3) (Fig. 6C).

There are a lot of interferents for NO detection in organisms. Some materialshave similar oxidation potential with NO, they may cause positive error; some bio-materials can induce electrode passivation through nonspecific adsorption, whichmay cause negative error. Therefore, good anti-interferent ability is essential. Theanti-interference ability of the microsensor was examined; results are shownin Table 2. The main interferents for NO detection in organisms, NO�

2 , DA, UA,and AA, have no interference (signal change <5%), indicating the good anti-interference performance of the microsensor.

Stability and reproducibility are key elements of electrode performance. Themicrosensor retained 90% of its initial response after it was kept in 0.1M phosphatebuffer solution (pH 7.4) for two weeks, which indicates their stability. The relativestandard deviations (RSD) for eight parallel measurements of 18 mM NO were4.4% for the same microsensor, and 8.9% for different microsensors. These resultssuggest that microsensors have good reproducibility.

Figure 6. A: Amperometric response of NO at the microsensor with successive injections of 1.8 mM NO

at the operational potential of 0.8V. B: Amperometric response of low concentrations of NO at the

microsensor. C: Detection limit of the microsensor.

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In organisms, NO is mainly biosynthesized via the catalysis of nitric oxidesynthase (NOS). With L-Arg and molecular oxygen as substrates, NOS catalyzes oneN atom from the two equivalent guanidine groups of L-Arg, forming L-citruline andreleasing NO (Wang and Marsden 1995). Constitutive NOS (cNOS) and inducibleNOS (iNOS) are the twomain kinds of NOS. There are plentiful iNOS inmacrophages.In 1985, Stuehr and Marletta first showed that macrophages can use its iNOS tobiosynthesize NO under the activation of lipopolysaccharide (LPS) and interferon-c(IFN-c) with L-Arg as precursor (Stuehr and Marletta 1985; Alessandro, Shinobu,and Luigi 1994). As a non-selective antagonist of NOS, NG-nitro-L-arginine(L-NNA) is able to inhibit the biosynthesis of NO (Rees et al. 1990; White et al.1993; Gardiner et al. 1990). In this system, we detected the NO released from macro-phages with the fabricated NO microsensor. The test was carried out in 5mL 0.1MPBS (pH 7.4) under stirring through amperometric measurements at 0.85V. WhenL-Arg was injected into the base solution containing macrophages activated by LPSand IFN-c, an apparent electrochemical signal belonging to the response of NOappeared. However, the electrochemical oxidation signal of NO would not be observedif macrophages were inhibited with L-NNA for 10min. These results were includedin Fig. 1, which indicates that our microsensors may have potential application valuein realizing the scatheless in vivo NO determination.

CONCLUSIONS

In this work, we realized the stable dispersion of CNFs in water through a novelnoncovalent method with the aid of AR. The resulting CNF-AR dispersion can bedirectly utilized for surface modification of carbon fiber microelectrodes. PAR-CNF

Table 2. Influence of some biomaterials on the peak current of

9.0� 10�5M NO

Interferents Concentration (M) Signal change (%)

glucose 9� 10�4 �1.89

caffein 9� 10�4 �3.84

DL-valine 9� 10�4 �3.98

L-glutamic acid 9� 10�4 þ2.76

L-aspartic acid 9� 10�4 �2.06

Glycine 9� 10�4 �1.03

cholesterol 9� 10�4 �1.73

L-serine 9� 10�4 �0.82

barbitone 9� 10�4 þ0.32

L-arginine 4.5� 10�4 þ2.08

L-tyrosine 4.5� 10�4 þ3.64

sucrose 4.5� 10�4 þ0.41

L-cystine 4.5� 10�4 þ1.62

epinephrine 9� 10�5 þ3.49

UA 9� 10�5 þ4.59

AA 9� 10�5 þ4.62

DA 9� 10�5 þ4.86

NO�2 9� 10�5 þ4.88

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composite films were fabricated through a simple and controllable electropoly-merization method. Surface characterization demonstrated that the PAR-CNFcomposite films had a three-dimensional and porous structure and an increasedsurface area. The PAR-CNF composite film has good electrocatalytic ability forthe oxidation of NO, which is useful for the fabrication of NO microsensor. Thefabricated NO microsensor had excellent performances and was applied in thedetection of NO released from macrophages successfully and indicates its potentialapplication value in the biomedical area.

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