COMMENT pubs.acs.org/ac
Response to the Comment on “Electrochemical Detection of Peroxynitrite Using a Biosensor Based on a Conducting PolymerManganese Ion Complex” Wei Choon Alvin Koh,† Jung Ik Son,† Eun Sang Choe,‡ and Yoon-Bo Shim*,† †
Department of Chemistry, Institute of BioPhysio Sensor Technology and ‡Department of Biological Sciences, Pusan National University, Busan 609-735, Korea
W
e hereby respond to Bedioui et al.’s comments regarding the lifetime of the target analyte ONOO at neutral pH. ONOO was introduced via a 0.1 mM donor solution of 3-morpholinosydnonimine (SIN-1). SIN-1 decomposes spontaneously in neutral aqueous media and consumes oxygen to release equimolar amounts of NO and superoxide simultaneously,16 which react rapidly to produce ONOO.7 Electron spin resonance spectroscopy (ESR) was used to derive the rate of ONOO formation at a speed of 1 μM/min from 1 mM SIN-1 present in phosphate buffer solution (PBS) which was used in the present study. In a previous report, SIN-1 was added to PBS (pH 7.4; 25 °C) in concentrations of 0.125, 0.25, 0.5, 1.0, and 2.0 mM. On the basis of the reported ESR of SIN-1 degradation and ONOO production rate, the concentrations of SIN-1 employed in the present study would yield 0.7560 μM ONOO total production.3 Although ONOO itself has a half-life of less than 1 s in an aqueous solution,8 the donor solution is relatively stable under these experimental conditions, having a half-life of 1426 min.7,9 This means that ONOO was constantly supplied for this amount of time. In the present study, SIN-1 was dissolved in solution and injected in the test solution after 30 min. Stable ONOO concentrations monitored by UVvis absorption at 302 nm have been reported.3,7 We confirmed the presence of ONOO in our experiments with UVvis spectroscopy (Figure 1), which shows the 302 nm absorption band of ONOO when SIN-1 was added to the solution. The inset of Figure 1 shows the time dependency of ONOO absorbance, showing that ONOO was produced continuously for more than 30 min. Liu et al. have shown that maximum ONOO production occurs when SIN-1 is left in an aqueous solution for at least 30 min.10 Since the reaction of NO and superoxide occurs virtually instantly, this period of time would maximize the effects of ONOO and limit the opportunity for NO and superoxide to exert any independent effects. To further confirm the response of ONOO, inhibitory experiments were performed using a chronoamperometric technique in the paper where a ONOO scavenger, such as uric acid, was added after four successive additions of ONOO standard solution (data not shown). The response current rose steeply and then arrived at an increased steady value after each addition of ONOO. However, upon addition of uric acid, the current response declined sharply to the baseline value. This is because ONOO was removed from the test solution almost immediately by uric acid. In this case, the potential difference between uric acid oxidation and ONOO reduction is high in our experimental conditions. In our case, we observed the uric acid oxidation peak at þ0.7 V. A similar anodic peak potential (Epa) for uric acid r 2011 American Chemical Society
Figure 1. UVvisible spectrum of ONOO at wavelength 302 nm. (Inset) Absorbancetime function graph of ONOO.
oxidation is þ0.76 V vs SCE at anodized electrodes.11 Since the reduction of ONOO is at þ0.2 V, there would be no uric acid oxidation to affect the ONOO current signal. Both experiments were performed in similar solution conditions. To answer the authors’ comments about Figure 4a,b, both parts a and b of Figure 4 show different experiments. In Figure 4a, ONOO was added to the test solution resulting in a spike in the response curve. There was some static charge in the case of Figure 4a when ONOO was added, which led to a sharp change in the current response where the applied potential was not changed. This spike was due to the sudden increase in static charge, and an equilibrium in nonfaradic current response was reached in about 15 s. The response time for detection was less than 1 s. On the other hand, there was no static charge in the case of Figure 4b when ONOO was added. ONOO was continuously produced by the SIN-1 donor solution for more than 20 min. We have stated this before in our response to reviewers’ comments previously. With regards to the authors’ comments on Figures 1b, 3, 4, and 5, cyclic voltammograms were recorded for the Mn-pDPB microelectrode from 0.2 to 0.6 V versus Ag/AgCl in 0.1 M PBS at pH 7.4. Although there are different conventions regarding the presentation of cyclic voltammograms (shown in Figure 1b),
Published: June 06, 2011 5465
dx.doi.org/10.1021/ac200710g | Anal. Chem. 2011, 83, 5465–5466
Analytical Chemistry
COMMENT
these are now commonly presented in Analytical Chemistry, according to IUPAC conventions, with oxidative current response as positive.12 Chronoamperometric experiments were performed by applying the reduction potential at the Mn-pDPB microelectrode to reduce ONOO. In the case of chronoamperometry, there was no IUPAC convention denoting this. Hence, we did not change the sign convention in Figures 3, 4, and 5 because the ONOO level was monitored through the change in reduction current of ONOO only. In addition, we would like to apologize for our mistake in the dynamic linear range for ONOO detection. It should be 20 nM30 μM. To answer the authors’ final comment, (i) the cyclic voltammograms in Figure 1b were obtained five times for repeated experiments, thus demonstrating their reproducibility. (ii) The measurements were made immediately after the addition of the aliquots. (iii) The cyclic voltammograms were recorded for the Mn-pDPB microelectrode from 0.2 to 0.6 V and then back to 0.2 V versus Ag/AgCl in 0.1 M PBS at pH 7.4. Thus, we confirm that the peroxynitrite biosensor is an effective tool for monitoring changes in in vitro extracellular peroxynitrite levels in response to stimulant drug exposure.
’ AUTHOR INFORMATION Corresponding Author
*Phone: (þ82) 51 510 2244. Fax: (þ82) 51 514 2430. E-mail:
[email protected].
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