A sensitive nitric oxide microsensor based on PBPB composite film-modified carbon fiber microelectrode

Sensors and Actuators B 133 (2008) 571–576

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

A sensitive nitric oxide microsensor based on PBPB composite film-modified carbon fiber microelectrode Yanfen Peng a,b , Chengguo Hu a , Dongyun Zheng a , Shengshui Hu a,b,∗ a b

College of Chemistry & Molecular Sciences, Wuhan University, Wuhan 430072, China State Key Laboratory of Transducer Technology, Chinese Academy of Sciences, Beijing 100080, China

a r t i c l e

i n f o

Article history: Received 2 February 2008 Received in revised form 20 March 2008 Accepted 21 March 2008 Available online 8 April 2008 Keywords: Carbon fiber electrode Nitric oxide Microsensor Electrochemistry

a b s t r a c t In this work, a sensitive electrochemical microsensor of nitric oxide (NO) was reported. The microsensor was constructed by coating PBPB composite on carbon fiber microelectrodes (CFME). The NO microsensor displayed excellent electrochemical activity toward the oxidation of NO and have the virtue of good stability, reproducibility and high sensitivity. Under optimal working conditions, the oxidation current of NO at this microsensor exhibited a good linear relationship with NO concentration in the range of 3.6 × 10−8 to 8.9 × 10−5 mol/L with a low detection limit of 3.6 × 10−9 mol/L (S/N = 3). The microsensor was successfully applied to the direct and real-time detection of NO release from biological samples, foreseeing the promising applications of this microsensor in fields like biology and medicine. © 2008 Elsevier B.V. All rights reserved.

1. Introduction The study of nitric oxide (NO) in biological systems has attracted considerable interest for decades, due to its important biological functions [1–3]. This reactive free radical formed from l-arginine is known to be the endothelium-derived relaxing factor [2], responsible for a number of diseases and a variety of physiological and pathological processes [4], such as a potent vasodilation [2], inhibition of platelet adhesion and activation [5], mediation of antitumor activities [6], and as an important neurotransmitter in the brain and peripheral nervous systems [7]. Techniques on accurate and rapid detection of NO are a significant subject in biomedical engineering and analytical chemistry. Whereas, the determination of NO generated from tissues faces great challenges because of the low concentrations of NO in biological samples and its short half-life due to autooxidation by reactions with endogenous oxygen, thiols, hemoglobin or other compounds to form nitrite or nitrite. A variety of methods are proposed for the determination of NO, including Griess assay, electron spin and paramagnetic resonance spectroscopy, chemiluminescence, UV–vis spectroscopy, fluorescence and electrochemical methods. Among these methods, electrochemical sensors and chemiluminescence were regarded to be capable of achieving the direct

∗ Corresponding author at: College of Chemistry & Molecular Sciences, Wuhan University, Wuhan 430072, China. Tel.: +86 27 8721 8904; fax: +86 27 6875 4067. E-mail address: [email protected] (S. Hu). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.03.036

and real-time measurement of NO [8,9]. Although the chemiluminescence provides high sensitivity, this method is expensive and complex, requiring the transfer of NO from solutions to the gas phases. Electrochemical sensors have been praised for its desirable advantages, for instance, the real-time, high-sensitive and direct analysis. They are also the only reliable analytical method to determine the in situ concentration of NO, without disturbing its metabolism and the associated regulatory pathways [10,11]. Sophie has reported to use an electrochemical sensor that allows the in vivo detection of NO in tumor-bearing mices for the first time [12]. Generally, two strategies are reported to achieve the sensitive determination of NO in vitro at carbon fiber microelectrodes (CFME). One is based on the direct oxidation of NO at electrodes coated with different membranes to cut-off interferents or substantially to reduce their contribution in the electrooxidation process, such as chloroprene, cellulose acetate [13] and WPI NO selective membranes [14]. The other involves the electrocatalytic oxidation of NO on carbon fiber electrodes modified with electropolymerized films of o-phenylenediamine [15], porphyrin [16] and eugenol [17], carbon nanotubes [18], and deposited o-phenylenediamine layers [19]. Of all these film materials, electropolymerized films of porphyrin are most extensively used. Conducing polymers have attracted much attention for promising applications in thin film transistors [20], polymer light-emitting diodes (LEDs) [21], electrochromic devices [22] and sensors. Thereinto, organic dye films are widely used as functional modifiers to catalyze the reduction or oxidation of biomolecules [23] as

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Scheme 1. Structural model of carbon fiber microelectrodes.

these materials can be easily synthesized from aqueous solutions and are stable in air and water [24]. Many reports have demonstrated that quinonimine dyes such as neutral red, toluidine blue, methylene blue, methylene green, and brilliant cresyl blue can be used for preparing chemically modified electrodes (CMEs) [23]. Bromophenol blue (BPB) is a triphenylmethane derivative and an effective electron redox mediator due to its conjugated system. To our knowledge, rare works deal with the determination of NO with this dye molecule. In this work, we reported a new method for preparing sensitive NO electrochemical microsensor by electropolymerizing PBPB composite (Fig. 1) and coating Nafion on a CFME. The Nafion/PBPB composite film-modified microelectrode (Nafion/PBPB/CFME) showed excellent electrocatalytic activity towards the oxidation of NO. The oxidation current linearly increased with NO concentration in the range of 3.6 × 10−8 to 8.9 × 10−5 mol/L with a low detection limit of 3.6 × 10−9 mol/L (S/N = 3). It is clear that the analytical performance of this NO microsensor is comparable or even better than most NO electrochemical sensors reported previously [15,29,14] (Table 1).

jing Reagent Factory, Beijing, China, and 5% Nafion solution (w/v), dopamine (DA) and ascorbic acid (AA) were the products of Sigma. All chemicals were of analytical grade and used as received. All solutions were prepared with double distilled water. High purity (99.999%) nitrogen gas was used for deaeration. Phosphate buffer solutions (PBS), containing 137 mmol/L NaCl, 2.7 mmol/L KCl, 8.0 mmol/L Na2 HPO4 and 1.5 mmol/L KH2 PO4 , were prepared with water and adjusted to pH 7.4 with 0.1 mol/L NaOH or 0.1 mol/L HCl. NO saturated aqueous solutions (at 20 ◦ C, [NO]≈1.8 mmol/L) were prepared by injecting 8 mL deoxygenated distilled water into a sealed bottle (250 mL) full of NO gas and keeping under NO atmosphere [25]. NO was generated by slowly dropping 6 mol/L H2 SO4 into a glass flask containing saturated NaNO2 solutions and formed in this disproportional reaction [26]. The generated gas was forced to pass through a 30% NaOH solution twice and water once to remove any NO2 − formed as a result of the reaction of NO with trace oxygen. Before the addition of H2 SO4 , all apparatus were degassed cautiously with nitrogen for 30 min to exclude O2 . Because NO gas is toxic at concentrations higher than 100 ppm, all the experiments were carried out in a fume hood. Addition of NO solutions during the electrochemical experiments was performed with a gas-tight microsyringe. Electrochemical experiments were performed with a CHI660A electrochemical workstation (CH Instruments, Shanghai, China) at room temperature. The three-electrode electrochemical cell was comprised of a saturated calomel reference electrode (SCE), a platinum wire counter electrode and a modified carbon fiber working electrode. In order to reduce surrounding electromagnetic interferences on the determination of NO, the electrochemical cell was hermetic and enclosed in a grounded Faraday cage.

2. Experiment

2.2. Fabrication of NO microelectrode

2.1. Reagents and chemicals

The CFME (Scheme 1) was prepared according to Wang’s work [18]. A carbon fiber (7.8 ␮m in diameter, Kureha Chemical Industry, Tokyo, Japan) was initially cleaned by sonicating in acetone, alcohol and doubly distilled water, each for 2 min. The carbon

Fig. 1. Structure of bromophenol blue.

Carbon fiber (7.8 ␮m diameter) was purchased from Kureha Chemical Industry, Tokyo, Japan. BPB was purchased from BeiTable 1 Comparison of detection limits, dynamic range and stability for different NO sensors Electrode CF ( = 30–35 ␮m, l = 100–150 ␮m) CF(=8 ␮m, lc = 1 mm) CF(d = 30-35 ␮m, l = 500 ␮m) CF( = 7 ␮m, l = 15 mm) CF( = 0.1 ␮m, l = 150 ␮m) CF(=6 ␮m, l = 2–4 mm) CF(=7.8 ␮m, l = 300–500 ␮m) a

a c b d e f i

CF = carbon fiber. l = length. o-PD = o-phenylenediamine. ␾ = diameter. m-PD = m-dihydroxybenzene. Resor = resorcinol. CA = cellulose acetate.

Modifiers

Sensitivity b

Nafion/o-PD Nafion/o-PD Nafion/m-PDe +resorf Nafion/WPI membrane WPI membrane CAi /Nafion PBPB composite film

31 pC/␮mol/L 9.6 nA/␮mol/L 0.5 nA/␮mol/L 1.03 pA/nmol/L 0.5 pA/nmol/L 0.44 nA/␮mol/L 1.04 pA/nmol/L

Detection limit 35 nmol/L N/A 60–80 nmol/L 5 nmol/L 2 nmol/L N/A 3.6 nmol/L

Dynamic range (mol/L) −7

−6

10 to 6 × 10 10−7 to 5 × 10−7 2 × 10−7 to 10−6 10−8 to 5 × 10−6 10−8 to 10−6 10−6 to 1.2 × 10−4 3.6 × 10−8 to 8.9 × 10−5

Ref. [15] [19] [29] [14] [30] [31] This work

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peak was also obtained at about +0.18 V. Due to the small surface area of CFME, the polymerism of BPB is less obvious than its electropolymerization on the glass carbon electrode [27]. The obtained PBPB/CFME was firstly washed with water and then immersed in 3% Nafion solution for three times (each for 10 s). The resulting Nafion/PBPB composite film CFME was donated as a NO microsensor. The microsensor was swept in the potential range of 0.3 to 1.1 V in PBS (pH 7.4) at a scan rate of 50 mV/s till stable cyclic voltammograms were obtained before use. 2.3. Procedures of electrochemical measurement

Fig. 2. Cyclic voltammograms of polymerization of BPB (0.1 mmol/L) in phosphate buffer solution (pH 5.6) at CFME in the potential range from −1.0 to 1.8 V at a scan rate of 50 mV/s. Inset: the cyclic voltammograms of circle portion are zoomed in.

fiber was cut into a desired length (cal. 2 cm) and connected to a copper wire for conductance by a silver glue. The carbon fibercopper wire conjugate was sealed in a capillary by the fusion of the microtip of the capillary on a glass lamp. The length of carbon fiber extended outside the glass capillary was about 1 cm. Following this, the protruded 1-cm carbon fiber was carefully etched on flame to produce a 300–500 ␮m carbon fiber, which was used as the active section of the microelectrode. Finally, the copper and the other end of the capillary were fixed with the silver glue. Through these processes, a needle-shaped carbon fiber microelectrode was obtained. The carbon fiber microelectrode was rinsed with redistilled water and underwent further surface treatments described below. Prior to electropolymerization, CFME was immersed in nitric acid for 15 s. Fig. 2 shows the continuous cyclic voltammograms of electropolymerization of BPB at CFME sweeping in the range of −1.0 to 1.8 V for 30 cycles at a scan rate of 50 mV/s in 0.1 mol/L phosphate buffer solution (pH 5.6) containing 0.1 mmol/L BPB component. An anodic peak at +0.74 V was observed due to the oxidation of hydroxy group of BPB monomer and a very little reversible redox

The electrocatalytic activity of PBPB/CFME toward NO was first tested by square-wave voltammetry in the range of 0.3 to 1.1 V in a deoxygenated PBS. Prior to NO measurements, the modified electrode was placed in a blank PBS (pH 7.4) and the potential was cycled between 0.3 and 1.1 V at a scan rate of 50 mV/s until a steady voltammetric response was obtained. Amperometric detection of NO was performed at a constant potential of 1.1 V under a stirred condition. After the baseline became stable, a certain volume of NO solution was added to the electrochemical cell containing 5 mLdeoxygenated PBS. 2.4. Detection of NO release from cavy kidney tissues Kidney tissue was prepared from adult cavy. First, placing the cavy (anesthetized with pentobarbital) on the dissection tray ventral side up, making an incision from the midsection. Second, cutting from the incision up to the lower jaw. Then both left and right kidneys were immediately excised and stored in refrigeratory on ice until they were used for experiments. Prior to NO measurement, the PBPB film CFME was also scanned between 0.3 and 1.1 V in a deaerated PBS until the stable response. Subsequent experiments probed the ability of the inserted microelectrode system to detect the presence of NO itself in the cavy kidney tissue as follows: clean the kidneys with PBS buffer solution (pH 7.4), utilize the copper wire through the cavy kidney tissue to fixup it on the beaker, then carve an aperture in the kidney; the microelectrode inserted to the aperture and clung to the kidney. Amperometric measurements were started under a stirred condition. During the experiment, 50 ␮L of 0.1 mol/L l-Arg was added into the PBS, and the amperometric curve was recorded continuously.

Fig. 3. SEM images of bare CFME (a and c) and PBPB/CFME (b).

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Fig. 4. Square wave voltammograms of the bare CFME (b) and the PBPB/CFME (a and c) with (a and b) and without 1.8 × 10−5 mol/L NO (c) in a deaerated phosphate buffer solution (pH 7.4).

3. Results and discussion 3.1. Surface morphology of NO microsensor Fig. 3 shows the scanning electron microscopic (SEM) images of a bare CFME and PBPB/CFME. It is clear that the surface of the carbon fiber is smooth and net (Fig. 3a). In contrast, the surface of PBPB/CFME is covered by a rough PBPB layer (Fig. 3b), demonstrating the successfully modification of carbon fibers by PBPB through electropolymerization. Fig. 3c shows the general view of a CFME, which indicates that the carbon fiber is tightly sealed in the glass capillary with a tip of 200 nm in diameter. Therefore, BPB composite can be coated on the CFME, producing PBPB/CFME with a large active surface for the high-sensitive determination of NO and a rather small dimension suitable for the in vivo measurements or single-cell analysis. 3.2. Electrochemical response of NO microsensor Fig. 4 shows the square wave voltammograms of the bare CFME (b) and the PBPB/CFME (a and c) with (a and b) and without 1.8 × 10−5 mol/L NO (c). At the bare CFME, only a small oxidation peak with a current of 1.549 nA at 0.752 V can be observed. At the PBPB/CFME, no signal is seen when NO was absent in the supporting electrolyte. However, when there was 1.8 × 10−5 mol/L NO in the supporting electrolyte, an apparent oxidation peak with a sensitive current of 21.22 nA at 0.748 V was obtained. Therefore, PBPB was able to promote the oxidation of NO and enhance the electrochemical response. According to Atta, the polymerization of dyes can form a cross-linked oligomer that decreases their proton-donor ability and leads to the enhancement of their electrocatalytic ability [28].

Fig. 5. Effect of the PBPB film thickness (cycles of electropolymerization) on the oxidation peak current of NO at the PBPB/CFME.

thickness at the nanoscale. Fig. 5 shows the influence of the cycle number in the polymerization process on the oxidation peak current of NO. The experimental results indicate that the sensitivity of the modified electrode toward NO increases with the number of the electropolymerization scans and reaches an optimal level at 30 scans. When the number of scan exceeds 30 cycles, the sensitivity decreases. Therefore, 30 cycles in the potential range of −1.0 to 1.8 V were chosen for the electropolymerization of BPB. 3.3.2. Influence of pH The optimization of the solution pH is very important for the electrochemical determination of NO. As the property of PBS is close to the nature environment of NO in vivo, we choose it as the supporting electrolyte. Fig. 6 shows the electrochemical behaviors of 1.8 × 10−5 mol/L NO at PBPB/CFME in PBS at different pH values. Although the oxidation potential of NO hardly changed with solution pH (data not shown), there is a considerable difference in the magnitude of the oxidation current. It can be seen that the peak current reached a maximum at pH 7.0. The oxidation of NO in solutions of pH 9.0 and pH 10.0 gave about the same current, but at pH 8.0 the current was lower than those obtained from other solutions. According to the biology environments, the determination pH value of NO was set at pH 7.4.

3.3. Optimization of experimental parameters 3.3.1. Optimization of the PBPB thickness The number of the electropolymerization scans of electropolymerization has a significant effect on the film thickness and the modifier amount of PBPB, which inevitably affects the determination of NO on PBPB/CFME. Thus, controlling the number of the electropolymerization scans of electropolymerization becomes a reproducible and readily adjustable procedure for modulating film

Fig. 6. Effect of solution pH on the oxidation peak current of 1.8 × 10−5 mol/L NO at PBPB/CFME by square wave voltammetry.

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Fig. 7. Amperometric response of NO microsensor with successive injections of different concentration of NO in a deaerated PBS (pH 7.4) at the operational potential of 1.1 V. Insert (a): the amplificatory images of amperometric response from 400 to 700 s. Insert (b): plot of the dependence of the measured current on NO concentrations. Insert (c): the amplificatory images of detection limit (S/N = 3).

3.4. Amperometric response of NO at the microsensor Under the optimized working conditions, amperometric measurement was carried out in a deaerated PBS. The whole cell was protected under nitrogen gas atmosphere during experiment. The potential of 1.1 V was applied to the Nafion/PBPB/CFME. Fig. 7 shows the amperometric response of NO microsensor when different concentrations of NO were added consecutively. The inset of Fig. 7a displays the magnification of the first three injections. As can be seen from Fig. 7, it displays a very fast response and a sharp increase in intensity immediately after each addition of NO. The signal is stable at the low concentration, but displays a relatively slow decrease when further increase the concentration. The shape of the amperometric signals is similar to typical amperometric responses related to other electrodes. The current response exhibits a very good linear relationship with the concentration of NO in the range of 3.6 × 10−8 to 8.9 × 10−5 mol/L (Fig. 7b), R2 = 0.9995. The sensitivity of this microsensor is 1.04 pA/(nmol L). The detection limit is estimated to be 3.6 × 10−9 mol/L (S/N = 3) (Fig. 7c). 3.5. Interference, stability and reproducibility One of the great obstacles for the practical applications of NO electrochemical sensors is the interference from species present in biomedical samples, such as nitrite, DA and AA. Here, the interferences of these substances on the determination of NO at this microsensor were examined. The results showed that no interference in the determination of 3.6 × 10−7 mol/L NO was observed when the recommended procedure was applied after the additions of 3.6 × 10−5 mol/L Na+ , K+ , Ca2+ , Mg2+ , Cu2+ , Cl− , NO3 − , SO4 2− , CO3 2− , PO4 3− , GOD, l-Arg, 1 × 10−5 mol/L NO2 − and 8.0 × 10−6 mol/L DA and AA. The stability of NO microsensor was investigated and the sensor was tested by amperometric response of 1.8 × 10−6 mol/L NO every 1–2 days. The response of sensor remained constant for the first 6 days, and remains 96% of its initial response after it was kept in air for 1 week. The reproducibility of NO microsensor was also tested. The relative standard deviation (R.S.D.) was 3.3% for six parallel detections of 1.8 × 10−5 mol/L NO at the same NO sensor. The amperometric responses of 1.8 × 10−6 mol/L NO at six different NO sensors had

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Fig. 8. Amperograms of NO microsensor in a deaerated PBS (pH 7.4) in the presence of cavy kidney tissue without adding 50 ␮L 0.1 mol/L l-Arg (a) and amperogram showing NO release from cavy kidney tissue by stimulation of 50 ␮L 0.1 mol/L l-Arg (b), both measured by the NO sensor. Applied potential: 1.1 V.

a R.S.D. of 7.2%, which suggests that this modified electrode has a good reproducibility for the determination of NO. 3.6. Measurement of NO release from cavy kidney tissue In biological systems, NO is biosynthesized from l-Arg via a specific biochemical pathway that includes nitric oxide synthase (NOS). Amperometric measurements at the operational potential of 1.1 V were carried out in a stirred deaerated 0.1 mol/L PBS. The result is shown in Fig. 8. When the stimulator, 50 ␮L 0.1 mol/L l-Arg was injected into 7.5 mL of 0.1 mol/L PBS (pH 7.4) without kidney, no current response appears (Fig. 8a). However, when 50 ␮L of 0.1 mol/L l-Arg was added into the PBS containing kidney, the amperometric current increased about 30 s later, which corresponds to the release of NO (Fig. 8b) [32], it also suggests that the microelectrode have practical applications as microsensor in NO monitoring system. 4. Conclusion In this paper, we prepared a sensitive NO microsensor based on the Nafion/PBPB/CFME, which was obtained by coating BPB composite on the surface of CFME. The NO microsensor could provide more active sites for the electrochemical oxidation of NO. The oxidation peak current of NO at the microsensor has a good linear relationship when the NO concentration is in the range of 3.6 × 10−8 to 8.9 × 10−5 mol/L. A low detection limit of 3.6 × 10−9 mol/L (S/N = 3) is obtained. This novel NO microsensor exhibits a high degree of stability, reproducibility and sensitivity and has been used for the determination of NO in the biomedical systems. Acknowledgements The authors acknowledge financial support by the National Nature Science Foundation of China (Nos. 60571042 and 30770549). References [1] L.J. Ignarro (Ed.), Nitric Oxide Biology and Pathology, Academic Press, San Diego, 2000. [2] L.J. Ignarro, G. Buga, K. Wood, R. Byrns, G. Chaudhuri, Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide, Proc. Natl. Acad. Sci. 84 (1987) 9265–9269.

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Biographies Yanfen Peng, is an MSc candidate in the College of Chemistry & Molecular Sciences, Wuhan University, PR China. Her current research is the fabrication of a nanosensor and its application in biological fields. Chengguo Hu, is a lecturer of the Department of Chemistry, Wuhan University, PR China. He received his Dr. degree in the College of Chemistry & Molecular Sciences, Wuhan University, PR China, in 2006. His current research is the bioelectroanalytical chemistry. Dongyun Zheng, is a PhD candidate in the College of Chemistry & Molecular Sciences, Wuhan University, PR China. Her current research is the study of nitric oxide biosensor based on nanomaterial. Shengshui Hu, is a professor of the Department of Chemistry, Wuhan University, PR China. From 1988 to 1990, he had been a guest research fellow in Chalmers University, Sweden. In 1995, he worked as a senior visiting scholar in Universita´ Degli Studi di Firenze, Italy. During 1995–1998, he was a visiting research fellow in Case Western Reserve University, USA.