A novel electrochemical sensor for nitric oxide using aligned RuO2 nanowires on a Pt filament

CY Wang1

, J Liu2, CA Piantadosi1, BW Allen1*

1 Departments of Medicine and Anesthesiology, Duke University Medical Center 2 Department of Chemistry, Duke University

* barry.w.allen@duke.edu

Abstract Nitric oxide (NO), has been found to play various key roles in many living organisms, since it was initially identified as the endothelial derived relaxing factor (EDRF) in 1987[6]. The great interest in nitric oxide research has created a

A novel electrochemical sensor was prepared for detecting nitric oxide (NO) in biological materials using aligned ruthenium oxide nanowires deposited on a Pt filament by chemical vapor deposition (CVD) in two steps. First, ruthenium carbonyl (Ru3(CO)12) was thermally decomposed in a quartz tube furnace, and a thin film of ruthenium oxide was allowed to condense on the inner wall of the tube in a peripheral region of the furnace. In the second step, the ruthenium film was oxidized at a higher temperature and allowed to diffuse away from the center of the furnace and to precipitate on the surface of a silicon wafer or Pt filament. X-Ray diffraction (XRD) and scanning electron microscopy (SEM) of the coated silicon wafer revealed that the product consisted of aligned ruthenium oxide (RuO2) nanowires. The nanowire-coated Pt filament was utilized as an amperometric electrochemical sensor to detect nitric oxide gas in the aqueous phase. This sensor had much higher sensitivity (specific current) to nitric oxide than bare Pt, and the detection limit was less than 1 nM. It is anticipated that this novel sensor can be used to make biological measurements in vivo.

demand for devices and methods which can make accurate measurements of nitric oxide in complex biological systems in order to unravel its actions. Especially important is the ability to study intercellular signaling by nitric oxide at physiological levels. Physiological levels are those needed to maintain normal function in an organism, tissue, cell or organelle that is free of injury or disease. These levels have been estimated to be in the low nanomolar and high picomolar range and are likely to be brief releases followed by rapid disappearance due to the presence of biological scavenging molecules. The half-life of nitric oxide in biological systems is in the range of a few seconds to a few tenths of a second. Thus, a useful sensor must have a short time constant in addition to excellent sensitivity. Methods[7,8] that have been used to measure nitric oxide, both in aqueous and gas phases, include bioassay, electron magnetic resonance, chemiluminescence, laser-induced fluorescence, spectrophotometry and electrochemistry. Optimal methods of detection should provide continuous monitoring of low level NO generation in real time and be applicable in vivo. Of the methods mentioned, however, electrochemistry is the most satisfactory since the others involve devices that are too large, too slow or too insensitive for real-time, in vivo measurement of nitric oxide at physiological levels. Several kinds of NO electrodes have been reported in literature and some have been commercialized [9]. The detection limits reported for these sensors range from low nanomolar to several tens of nanomolar.

Keywords: sensor, nitric oxide, aligned, RuO2, nanowire

Introduction With the development of nanotechnology, nanomaterials have attracted the attention of scientists in fields such as optics and electronics. Nanomaterials have an important application in the development of new gas sensors, due to their novel properties. Thin films assembled from nanoscale particles or from nonporous materials have been used as sensor elements [1,2], as have so-called one-dimensional nanomaterials such as carbon nanotubes, silicon nanowires and semiconductor nanoribbons [3-5].

Detection limits in the low nanomolar range have been reported for a ruthenium (Ru) working electrode, in which an electrochemical conditioning technique was used to produce

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multiple layers of ruthenium oxide (RuOx) on the Ru surface [10,11]. But Ru filaments small enough to be suitable for biological applications are not commercially available, because the extreme hardness and high melting point of the metal make it very difficult to work. Therefore, we resolved to experiment with the deposition of Ru on filaments made of more tractable metals. In addition, we hoped to exploit the unique surface properties of nanoscale materials, such high surface-to-volume ratios, which have previously shown promise for gas sensors.

Experimental Synthesis of RuO2 nanowires Ru carbonyl, (Ru3(CO)12), was placed in an alumina combustion boat at the center of a quartz tube furnace and heated to150°C for 1 hour in a flowing argon atmosphere, and ruthenium oxide was allowed to condense in a cooler region of the tube. The furnace was then shut off. In a second step, the quartz tube was moved so as to center the condensed oxide film within the heating elements of the furnace. Either a piece of Si wafer (coated with a SiO2 layer of 500 nm) or a platinum wire (0.76 mm in diameter) was placed in the quartz tube away from the heating elements, and the temperature was maintained at 1000° C in an atmosphere of still air for 4-5hrs. The morphology and composition of the product obtained on Si wafer were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM,). The coated Pt wire was used to make the NO-selective electrode (see below). Preparation of NO working electrode: One end of the 76 µm diameter Pt filament coated with ruthenium oxide served as the working electrode. Ag/AgCl was used as reference electrode and bare Pt was used as auxiliary electrode. Testing the response to NO The response of the working electrode to nitric oxide was tested as previously described [10,11]. at 35º C using a potentiostat ( Model BAS 100B) equipped with a preamplifier. The electrolyte was phosphate-buffered saline (PBS) at pH 7.0 phosphate buffer (Fisher). The electrode was preconditioned for 20mins by potential cycling between +200 and +800 mV and then conditioned for 2hrs at a constant potential of +675 mV. Then the electrolyte was deaerated by bubbling with N2 for 30min. Detection of NO oxidation current was

performed at +675mV. Different microliter volumes of PBS saturated with 0.1% NO in N2. were injected into the PBS bathing the electrodes.

Results and discussion We checked the product on the silicon wafer by X-ray diffraction, which illustrates that the product is RuO2. Fig.1a shows an SEM image of the product on silicon wafer. It can be seen that the RuO2 is wire-like with diameters of 80±10nm,

A

B

C

Fig.1 SEM images of the product on silicon wafer (A) and on 76 µm diameter Pt filament (B and C). The RuO2 nanowires were aligned with each other due to the high density.

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and the nanowires are aligned due to their high density.

Fig. 2 shows the response of the electrode to nitric oxide: different volumes of PBS saturated with 0.1%NO were injected into a 2-ml cell.

In order to study the morphology of the Ru oxide on the actual Pt working electrode, SEMs shown in Figs.1B and.1C were obtained, which reveal that the nanowires stand up on the Pt substrate.

The injection of 2 µl. of the NO solution produces a clear signal. The solubility of NO in PBS at room temperature and 37º C are 1.9 and 1.4 mM atm-1 respectively. Therefore, when 2-µl saturated 0.1% NO PBS solution is injected into 2ml PBS at room temperature, the true detected concentration of NO is less than 1.9nM. Since the 2-µl saturated 0.1%NO PBS solution at room temperature was injected into 37°C solution in our experiment, and the test system was not sealed, the real concentration of NO that was detected should be considerably less than 1.9nM considering that some of NO goes out of the solution at its diffusion rate of 50 µm/s before it was detected by the working electrode.

From Fig.1B we estimate that the average length of the RuO2 is around 650nm, since the diameter of the Pt filament coated with RuO2 is 77.3 um and the diameter of commercial Pt filament is 76 um. A vapor-liquid-solid (VLS) mechanism may explain the formation of aligned RuO2 nanowires by this two-step deposition: first step might be the decomposition of ruthenium carbonyl and the precipitation of a ruthenium film on the surface of quartz tube at low temperature in the Ar atmosphere. In the second step the ruthenium is oxidized at high temperature, forming ruthenium oxide nanowires in a cooler region of the furnace in air by a VLS mechanism. The first step at low temperature, which results in slow evaporation and uniform deposition, is necessary for the formation of aligned nanowires. If, however, the ruthenium carbonyl is decomposed directly in air at high temperature, the ruthenium oxide will deposit on the Pt wire not as nanowires but as a thin, powdery film which is poorly adherent.

Conclusions: Aligned ruthenium oxide nanowires were electrochemically sensitive to nitric oxide, and can detect the nitric oxide in low nanomolar concentrations. The small diameter and low detection limit of these electrodes make them suitable for in vivo evaluation.

Acknowledgements

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This work was supported by a startup fund from Duke University Department of Chemistry and by the Air Force Office of Scientific Research (to J.L.) and by the Office of Naval Research to (B.W.A. and C.A.P.)

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Fig.2 The amperometric response of the electrode is shown to injections of three test solutions in a 2 mL volume of stirred PBS maintained at 35ºC. (NO oxidation currents are in the downward direction) . At the first arrow 5µL of a control solution (N2-saturated PBS) was injected. At the second and third arrows 5µl and 2µL, respectively, of PBS saturated with 0.1%NO were injected. The 2µL injection corresponds to 1.9nM NO.

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