Technical Note pubs.acs.org/ac
Improved Electrochemical Microsensor for the Real-Time Simultaneous Analysis of Endogenous Nitric Oxide and Carbon Monoxide Generation Sarah S. Park,† Jiyeon Kim,‡ and Youngmi Lee*,† †
Department of Chemistry and Nano Science, Ewha Womans University, Seoul, 120-750, Korea Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600
‡
S Supporting Information *
ABSTRACT: An amperometric dual NO/CO microsensor was developed on the basis of a working electrode incorporating dual Pt microdisks (each diameter, 76 μm) and a Ag/AgCl reference electrode covered with a gas permeable membrane. One of the Pt disks was sequentially electrodeposited with Pt and Sn; the other Pt disk was deposited with Pt−Fe(III) oxide nanocomposites. The first showed activity for the oxidation of both NO and CO; the second showed activity only for NO oxidation. In the copresence of NO and CO, the currents measured at each electrode, respectively, represented the concentrations of CO and NO. The sensor showed high stability during the monitoring of organ tissue for at least 2.5 h and high selectivity to NO over CO at the Pt−Fe(III) oxide working electrode. Real-time coupled dynamic changes of NO and CO generated by a living C57 mouse kidney were monitored simultaneously and quantitatively in response to a NO synthase inhibitor (NG-nitro-L-arginine methyl ester), for the first time. CO was found to increase and NO decreased upon addition of the inhibitor, suggesting a possible reciprocal interaction between these endogenous gases.
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iatomic nitric oxide (NO)1−3 and carbon monoxide (CO)4−6 play diverse biological and physiological roles as signaling molecules; they have many common properties. They share primary molecular targets (e.g., soluble guanylate cyclase) and play similar biological roles (e.g., vasodilatation, inhibition of platelet aggregation, anti-inflammatory activity, and neurotransmission). Their generation systems are also similar: NO is generated endogenously via NO synthase (NOS) isozymes from L-arginine, and CO is generated via heme oxygenase (HO) isozymes from heme. Both enzyme families require oxygen and a reducing agent, NADPH, for their activity. NO and CO gases interact with each other, complicating their behaviors. Their interactions have been suggested to be either synergic or antagonistic depending on their relative concentrations,7 though the exact relationship between NO and CO is not fully understood. Most studies on the interactions between NO and CO have relied on post-mortem immunohistochemical analysis of the expression of the corresponding gas-generating enzymes. The real-time simultaneous quantitative measurement of NO and CO would aid understanding of their complex interactions in both physiological and pathologic systems relevant to many diseases. The detection of these endogenous gases in biological systems is difficult because they are high diffusible, decay rapidly (via reactions with oxygen, heme, etc.), and exist at low concentrations. NO measurements have been carried out mainly via spectroscopy and using electrochemistry. A variety of electrochemical NO sensors have been developed and are widely used because of their attractive capability of direct, fast analysis © 2012 American Chemical Society
with high sensitivity.8 The measurement of CO in biological systems has been attempted indirectly via methods such as gas chromatography coupled to suitable detection methods (e.g., reduction gas detection,9 mass spectrometry10), radioisotope counting,11,12 or UV−visible spectroscopy analysis for CObound protein.13,14 A rather direct measurement for real-time detection of CO generated from vascular cells has been reported via mid-IR laser spectroscopy.15,16 However, real-time in vivo direct measurements of CO are not currently available. The simultaneous detection of NO and CO in biological systems is also undeveloped. A recently reported real-time concurrent analysis of NO and CO employed an amperometric dual microsensor and could measure both gases at mouse kidneys,17 though it required further improvement, particularly in terms of stability since it lost much sensitivity after prolonged measurement. This work reports the development of an improved NO/CO dual microsensor which has one sensing electrode codeposited with Pt and Fe(III) oxide nanoparticles and another sequentially electrodeposited with Pt and Sn. The high catalytic activity of Pt−Fe(III) nanoparticle composites for electrochemical oxidation of NO was reported by Xiangqin Lin et al.18 In fact, the newly optimized dual sensor could simultaneously measure surface concentrations of NO and CO generated from Received: November 29, 2011 Accepted: January 3, 2012 Published: January 20, 2012 1792
dx.doi.org/10.1021/ac2031628 | Anal. Chem. 2012, 84, 1792−1796
Analytical Chemistry
Technical Note
the kidneys had their outermost membranes removed, were cleaned with PBS solution (pH = 7.4), and then were immersed in PBS. The sensor was positioned 10 μm above the PBS bathed kidneys’ surfaces using a micromanipulator (World Precision Instrumentation Inc. Sarasota, FL). After the amperometric signals corresponding to NO and CO concentrations reached steady-state levels (∼10 min), 10 mM L-NAME was added to the buffer and the signal changes were monitored for ∼2.5 h. Currents at the dual sensor were recorded concurrently using a CHI900B bipotentiostat and converted to gas concentrations using the prior calibration curves acquired immediately before the tissue experiments.
living mouse (C57) kidney tissue with improved stability and accuracy. Meausurements in the presence of NG-nitroL-arginine methyl ester (L-NAME, a NOS inhibitor) demonstrated the dynamic and close correlation between endogenous NO and CO in the kidney tissue.
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EXPERIMENTAL SECTION Chemicals and Materials. Pt wire (diameter, 76 μm) and Ag wire (diameter, 127 μm) were from Good Fellow (Huntingdon, England). Theta type glass capillaries (diameter, 1.5 mm) were from World Precision Instrumentation Inc. (Sarasota, FL). Expanded poly(tetrafluoroethylene) (ePTFE) membrane was from W. L. Gore and Associates (Elkton, MD). Argon (Ar), NO, and CO gases were from Dong-A Gas Co. (Korea). H2SO4, NaCl, SnCl4, and phosphate buffered saline (PBS) were from Fisher Scientific (Rochester, NY). Chloroplatinic acid (8% and 3%) in water was from Sigma-Aldrich (St. Louis, MO) and LabChem Inc. (Pittsburgh, PA), respectively. FeCl3, and L-NAME were from Sigma-Aldrich (St. Louis, MO). All solutions were prepared with 18 MΩ·cm deionized water using reagent grade compounds without further purification. Preparation of NO/CO Dual Sensors. The dual NO/CO sensor was prepared similarly to that previously reported.17 First, the two bare Pt disks of the glass-sealed dual working electrode (WE, Pt disks’ diameters, 76 μm and separation, ∼85% (10 h experiment) of the initial one) compared with the previously reported sensor based on Pt/Sn (WE1) and Pt (WE2), which showed great sensitivity losses to
